Means and methods for detecting methylated dna

ABSTRACT

The present application relates to a nucleic acid molecule having a nucleotide sequence encoding a bifunctional polypeptide comprising the DNA-binding domain of a protein belonging to the family of Methyl-CpG binding proteins (MBDs) and the Fc portion of an antibody. In addition, vectors and host cells which comprise said nucleic acid molecule and polypeptides which are encoded by said nucleic acid molecule as well as processes for producing said polypeptide are disclosed. Moreover, the present application provides an antibody specifically binding said polypeptide and compositions, in particular diagnostic compositions comprising the nucleic acid molecule(s), vector(s), host cell(s), polypeptide(s) or antibodie(s) of the present application. Furthermore, methods and uses employing the polypeptides of the present invention for detecting methylated DNA, in particular in tumorous tissue or tumor cells are provided.

RELATED PATENT APPLICATIONS

This application is a continuation of U.S. patent application Ser. No.11/569,051 filed Nov. 13, 2006, entitled “MEANS AND METHODS FORDETECTING METHYLATED DNA”, naming Michael Rehli as inventor which is anational stage of International Patent Application PCT/EP2005/12707filed on Nov. 28, 2005 entitled “MEANS AND METHODS FOR DETECTINGMETHYLATED DNA”, naming Michael Rehli as applicant and inventor whichclaims the benefit of EP 04 02 8267.5 filed Nov. 29, 2004 entitled“MEANS AND METHODS FOR DETECTING METHYLATED DNA”, naming Michael Rehlias inventor. The entire content of the foreign patent applications areincorporated herein by reference, including, without limitation, alltext, tables, and drawings.

The present application relates to a nucleic acid molecule having anucleotide sequence encoding a bifunctional polypeptide comprising theDNA-binding domain of a protein belonging to the family of Methyl-CpGbinding proteins (MBDs) and the Fc portion of an antibody. In addition,vectors and host cells which comprise said nucleic acid molecule andpolypeptide which are encoded by said nucleic acid molecule as well asprocesses for producing said polypeptides are disclosed. Moreover, thepresent application provides an antibody specifically binding saidpolypeptide and compositions, in particular diagnostic compositionscomprising the nucleic acid molecule(s), vector(s), host cell(s),polypeptide(s) or antibodie(s) of the present application. Furthermore,methods and uses employing the polypeptides of the present invention fordetecting methylated DNA, in particular in tumorous tissue or tumorcells are provided.

The information to make the cells of all living organisms is containedin their DNA. DNA is made from 4 bases abbreviated as G, A, T, and C,and is built like a very long ladder with pairs of these letter makingup each of the “rungs” of the ladder. The letter G pairs with C and Awith T. Strings of these pairs store information like a coded message,with the information to make specific molecules grouped into regionscalled genes. Every cell of diploid animals contains two copies of everyone of our genes, with one copy of each gene coming from the mother andone copy from the father. (The only exceptions to this rule are genes onchromosomes that determine whether organisms develop as a “male” or a“female”.)

DNA Methylation and Gene Regulation

Apart from the four bases—adenine, guanine, cytosine and thymine—that“spell” our genome, there also is a fifth base which is produced by themodification of the post-replicative DNA. DNA methyl transferases(DNMTs) can catalyse the transfer of a methyl group from the methyldonor S-adenosylmethionine to the cytosine ring, and thereby produce thebase 5-methylcytosine. Specific cytosine residues are modified inmammals, which precede a guanosine residue in the DNA sequence (CpGdinucleotide) (Singal, Blood 93 (1999), 4059-4070); Robertson, Nat. Rev.Genet. 1 (2000), 11-19; Ng, Curr. Opin. Genet. Dev. (2000), 158-163;Razin, EMBO J. 17 (1998), 4905-4908). The methylation of CpGdinucleotides generally correlates with stable transcriptionalrepression and presumably leads to the fact that large parts of thenon-coding genome and potentially harmful sequences such as transposons,repeats or viral inserts are not transcribed. It is interesting that CpGdinucleotides are very unevenly distributed in the genome (Singal(1999), loc. cit., Robertson (2000), loc. cit., Ng (2000), loc. cit.,Razin (1998), loc. cit.). A large part of the genome contains much fewerCpGs than is statistically expected. This is presumably due to the factthat 5-methylcytosine deaminates comparatively easily to thymidine,which, in the course of evolution, leads to a relative decrease in thenumber of CpG dinucleotides. There are, however, again and again, largernumbers of CpGs distributed within the genome, so-called CpG islands.These regions often contain transcription initiation points and genepromoters and are generally not methylated in contrast to the CpGs whichare not associated with CpG islands. In normal cells, the methylation ofCpG islands has been observed only in exceptional cases such as theinactivation of the second copy of the x-chromosome in female cells andthe parental imprinting genome (Singal (1999), loc. cit., Robertson(2000), loc. cit., Ng (2000), loc. cit., Razin (1998), loc. cit.).

Regulation of DNA Methylation

It is only partly understood how DNA methylation patterns areestablished in the course of the embryogenesis and how the CpGmethylation is maintained and regulated in the genome (Singal (1999),loc. cit., Ng (2000), loc. cit., Razin (1998), loc. cit.). In mammalspecies, there are three DNA methyl transferases known (DNMT1, 3a and3b) which catalyse the DNA methylation process. The corresponding sharethat each DNMT contributes to the maintenance and regulation of the CpGmethylation must, however, still be clarified. Yet, all three enzymesare obviously essential to embryogenesis, the corresponding knockoutmice die in utero or shortly after birth (Bestor, Hum. Mol. Genet. 9(2000), 2395-2402; El Osta, Bioessays 25 (2003), 1071-1084). In themeantime, the connection between DNA methylation, modifications of thechromatin structure and certain histone modifications has been shownseveral times. The methylation of DNA mostly correlates with histonedeacetylation and methylation of the lysine 9 residue at histone H3(Sims, Trends Genet. 19 (2003), 629-639, Fahrner, Cancer Res. 62 (2002),7213-7218). Accordingly, DNMTs are associated with histone acetylases(HDACs) or co-repressor complexes. It is also hardly known how methylgroups are removed from CpG residues. In proliferating cells, the DNAmethylation can probably also take place passively during replication.There are, however, also examples of DNA demethylation in post-mitototiccells which can be explained by the existence of an active, yet unknowndemethylase (Wolfe, Proc. Natl. Acad. Sci. 96 (1999), 5894-5896).

CpG Methylation and Gene Silencing

Methylation of promoters (but not of non-regulating sequences)correlates with stable, transcriptional repression (Singal (1999), loc.cit., Ng (2000), loc. cit., Razin (1998), loc. cit.). The repressiveproperties of 5-methylcytosine can be mediated by two mechanisms.Firstly, the DNA methylation can directly impair the binding oftranscription factors. The second possibility, which is likely to beresponsible for the largest part of repression, is the recruitment ofmethyl-CpG-binding proteins (MBPs) (Ballestar, Eur. J. Biochem. 268(2001), 1-6). MBPs such as MECP2 or MBD2 (a component of the MeCP1complex) are accompanied by co-repressor complexes and HDACs which havea repressive effect and are responsible for the formation of densechromatin structures inaccessible to transcription factors(heterochromatin) (Ballestar (2001), loc. cit.).

Epigenetic Changes in Tumorigenesis

It keeps becoming clearer that the formation of tumours is supported notonly by genetic lesions (e.g. mutations or translocations) but also byepigenetic changes. An abnormal chromatin structure or DNA methylationcan influence the transcriptional status of oncogenes or tumoursuppressor genes and can promote tumour growth. Changes in the DNAmethylation include either the loss of methylation in normallymethylated sequences (hypomethylation) or the methylation of normallyunmethylated sequences (hypermethylation) (Roberston (2000), loc. cit.,Herman, N. Engl. J. Med. 349 (2003), 2042-2054; Momparler, Oncogene 22(2003), 6479-6483; Esteller, Science 297 (2002), 1807-1808; Plass, Hum.Mol. Genet 11 (2002), 2479-2488).

Hypomethylation

A global DNA hypomethylation has been described for almost all kinds oftumours. In tumour tissue, the content in 5-methylcytosine is reducedcompared to normal tissue with the major share of demethylation eventsbeing found in repetitive satellite sequences or in centromer regions ofthe chromosomes. However, in single cases, the demethylation andactivation of proto-oncogenes such as, e.g., bcl-2 or c-myc have alsobeen described (Costello, J. Med. Genet. 38 (2001), 285-303).

Hypermethylation of CpG Islands

CpG islands in general exert gene regulatory functions. This is why achange in the status of methylation correlates mostly directly with achange in the transcriptional activity of the locus concerned (Robertson(1999); Herman (2003); Esteller (2002); Momparler (2003); Plass (2002),all loc. cit.). Most CpG islands are present in unmethylated form innormal cells. In certain situations, CpG islands can, however, also bemethylated in gene regulatory events. The majority of CpG islands of theinactivated X-chromosome of a female cell are, for example, methylated(Goto, Microbiol. Mol. Biol. Rev. 62 (1998), 362-378). CpG islands canbe methylated also in the course of normal aging processes (Issa, Clin.Immunol. 109 (2003), 103-108).

It is in particular in tumours that CpG islands which are normally notmethylated can be present in a hypermethylated form. In many cases,genes affected by the hypermethylation encode proteins which counteractthe growth of a tumour such as, e.g., tumour suppressor genes. Thefollowing Table lists examples of genes for which it could be shown thatthey can be inactivated in tumours through the epigenetic mechanism ofhypermethylation.

TABLE Hypermethylated genes in tumours (examples) gene chromosomefunction cell cycle p16 9p21 cycline-dependent kinase control inhibitorp15 9p21 cycline-dependent kinase inhibitor Rb 13q14 cell cycleinhibition p73 1p36 p53-like protein DNA repair MLH1 3p21 DNA mismatchrepair protein GSTPI 11q13 inhibitor of oxidative DNA damage O6-MGMT10q26 DNA methyltransferase BRCA1 17q21 DNA repair protein apoptosisTMS-1/ASC 16p12-p11 adaptor for caspase 1 caspase 8 2q33-q34 PCDinitiator (Fas, Trail, TNF, . . .) DAPK1 9q34 PCD by IFNγ invasion/E-cadherin 16q22 adhesion molecule architecture VHL 3p26-p25angiogenesis-promoting protein TIMP-3 22q12-q13 metalloproteinaseinhibitor THBS1 15q15 angiogenesis inhibitor growth ER-α 6q25 estrogenreceptor factor RAR-β 3p24 retinoic acid receptor response SOCS-1 16p13neg. regulator in the JAK/STAT signal path

Reasons for the tumour-specific hypermethylation are almost unknown.Interestingly, certain kinds of tumours seem to have their ownhypermethylation profiles. It could be shown in larger comparativestudies that hypermethylation is not evenly distributed but that itoccurs depending on the tumour. In cases of leukaemia, mostly othergenes are hypermethylated compared to, for instance, colon carcinomas orgliomas. Thus, hypermethylation could be useful for classifying tumours(Esteller, Cancer Res. 61 (2001), 3225-3229; Costello, Nat. Genet. 24(2000), 132-138).

In many cases, hypermethylation is also combined with an increasedactivity of HDACs. After treatment with demethylated substances (e.g.5-azacytidine), methylated genes could only be reactivated after alsousing HDAC inhibitors (such as, e.g., trichostatin A (TSA)) (Suzuki,Nat. Genet. 31 (2002), 141-149; Ghoshal, Mol. Cell. Biol. 22 (2002),8302-8319; Kalebic, Ann. N.Y. Acad. Sci 983 (2003), 278-285).

Most analyses suggest that the DNA methylation is dominantly repressedand that it cannot be reversed by a treatment with HDAC inhibitors suchas TSA (Suzuki (2002); Ghoshal (2002), loc. cit.). There are, however,also more recent indications that valproate, a HDAC inhibitor which isalready used in clinics, can lead to the demethylation of DNA (Detich,J. Biol. Chem. 278 (2003), 27586-27592). However, no systematic analyseshave so far been carried out in this respect.

Clinical Approaches for Reversing Epigenetic Changes

While genetic causes of cancer (such as, e.g., mutations) areirreversible, epigenetic changes contributing their share to thetumorigenesis might possibly be reversible. Thus, the possible treatmentof epitgenetic changes offers new possibilities of therapy for thetreatment of neoplasias (Herman (2003); Momparler (2003); Plass (2002),all loc. cit.; Leone, Clin. Immunol. 109 (2003), 89-102; Claus, Oncogene22 (2003), 6489-6496).

More than 20 years ago, 5-azacytidine has already been developed as ananti-neoplastic medicament and used without the molecular effect of thesubstance being known. Nowadays, it is already used successfully in afurther developed form (Deoxy-5-azacytidine, Decitabine) for thetreatment of myelodysplastic syndromes and secondary leukaemia (Leone(2003), loc. cit.; Lyons, Curr. Opin. Investig. Drugs 4 (2003),1442-1450; Issa, Curr. Opin. Oncol. 15 (2003), 446-451). Due to the invitro observation that HDAC inhibitors can support the reactivation ofmethylated promoters and can act synergistically with demethylatedsubstances, at present pilot studies are carried out throughout theworld, combining the use of both classes of substances (Kalebic (2003);Claus (2003), loc. cit.; Gagnon, Anticancer Drugs 14 (2003), 193-202;Shaker, Leuk. Res. 27 (2003), 437-444).

Detection Methods for the Analysis of CpG Methylation

The development of detection methods for the analysis of genomic CpGmethylation has mainly gained importance due to the fact that it hasbeen found that changes in the CpG methylation pattern can be associatedwith diseases such as cancer. At present, there are mainly techniquesknown which are used for the detection of the CpG methylation of knowngene loci (Dahl, Biogerontology 4 (2003), 233-250). Methods allowing ananalysis of the CpG methylation throughout the genome are lessestablished. In the following, the most common methods for analysis ofCpG methylation together with their main fields of application aresummarised.

Use of Methylation-Sensitive Restriction Enzymes for the Detection ofCpG Methylation

The methylation status of specific CpG dinucleotides can be determinedusing isoschizomers of bacterial restriction endonucleases which arecharacterised by different sensitivities vis-à-vis 5-methylcytosine.Examples thereof are the enzymes HpaII and MspI—both cut CCGG sequences,HpaII however only if the internal cytosine is not methylated. Someassays are based on the use of methylation-sensitive restrictionenzymes, said assays being used for both the analysis of individualgenes and analysis of the CpG methylation throughout the genome. Thefragments of a methylation-sensitive restriction digestion are mostlydetected by means of Southern blot or a genomic PCR of the regionflanking the restriction site (Dahl (2003), loc. cit.). All analyses ofthe CpG methylation throughout the genome, which have been published upto today, use methylation-sensitive restriction enzymes as a componentof the method. Restriction Landmark Genomic Scanning (RLGS) (Costello,Methods 27 (2002), 144-149), for instance, uses a kind oftwo-dimensional agarose gel electrophorese in which every dimension isdigested with a different methylation-sensitive restriction enzyme toidentify differences in the CpG methylation of two DNA populations.Methylated CpG Island Amplification (MCA) enriches fragments withmethylated SmaI restriction sites and uses LM-PCR for enriching thefragments. Such amplification products have already been successfullyanalysed by means of Representational Difference Analysis (RDA) (Smith,Genome Res. 13 (2003), 558-569) or CpG island microarrays (Yan, CancerRes. 6 (2001), 8375-8380).

With regard to the analysis of the CpG methylation throughout thegenome, all assays that are based on methylation-sensitive restrictionenzymes have disadvantages. In order to carry out the assays in anoptimal way, it has, amongst others, to be guaranteed that allrestriction digestions are completed. The greatest disadvantage is thatthe analyses merely inform on the methylation status of the cytosineresidues which have been recognised by the methylation-sensitiverestriction enzymes used. The selection of the restriction enzymesautomatically limits the number of detectable sequences—a neutralanalysis of the CpG methylation is therefore not possible.

Bisulfate Treatment for the Analysis of the CpG Methylation

The treatment of double-stranded genomic DNA with sodium bisulfate leadsto the deamination of unmethylated cytosine residues into uracilresidues and to the formation of two single strands that are no longercomplementary. During this treatment, 5-methyl cytosine is maintained.The differences in sequence produced in this way form the basis of thedifferentiation between methylated and unmethylated DNA (Frommer, Proc.Natl. Acad. Sci. 889 (1992), 1827-1831). DNA treated with bisulfite canbe used directly in PCR in which uracil residues (previouslyunmethylated cytosine) and thymidine residues are amplified as thymidineand only 5-methylcytosine residues are amplified as cytosine residues.Depending on the application, the primers used for the PCR differentiatebetween methylated and unmethylated sequences or amplify fragmentsindependently of the methylation status. PCR fragments which have beenamplified using non-discriminating primers can, for instance, besequenced directly to determine the share in methylated and unmethylatedCpGs. Further methods make use of the physical differences of such PCRfragments (melting behaviour, single-strand conformation, restrictionsites for restriction enzymes, etc.) for determining the degree ofmethylation (Dahl (2003), loc. cit.). Other methodical approachesutilise the differences in sequence for the specific amplification ofmethylated and unmethylated sequences by discriminating primers orprobes (methylation-specific PCR, methylight PCR) (Dahl (2003), loc.cit.). Bisulfite-inducing differences in sequence of PCR products canalso be found by means of methylation-specific oligonucleotide (MSO)micro-arrays (Shi, J. Cell. Biochem. 88 (2003), 138-143; Adorjan,Nucleic Acid Res. 30 (2002), e21; Gitan, Genome Res. 12 (2002),158-164).

In contrast to the methylation-sensitive restriction enzymes, the DNAtreated with bisulfite can provide information on the methylation statusof several CpG residues in an amplified genomic fragment. The treatedDNA is not suitable for analyses throughout the genome presumably due toits reduced complexity and its high degree of denaturation.

Further Methods for the Detection of CpG Methylation

Antibodies against 5-methyl cytosine recognise CpG methylation indenatured, single-stranded DNA are used mainly for theimmunohistochemical staining of the CpG methylation on the chromosomesof individual, fixed cells. Yet, these antibodies are not suitable forenriching methylated sequences.

Already in 1994, the laboratory of A. Bird developed a method forenriching methylated DNA fragments by means of affinity chromatography(Cross, Nat. Genet. 6 (1994), 236-244). A recombinant MECP2 bound to amatrix was used for binding the methylated DNA. Since then thistechnique has been used, improved and combined with further techniquesby other working groups (Shiraishi, Proc. Natl. Acad. Sci. 96 (1999),2913-2918; Brock, Nucleic Acid. Res. 29 (2001), E123). The binding ofstrongly or less strongly methylated genomic sequences to an affinitymatrix depends on the salt concentration which makes it possible toseparate the CpG islands with dense methylation from other sequenceswith a lower methylation density. The disadvantage of this affinitychromatography is the large amount of genomic DNA required (50-100 μg)and the relatively time-consuming procedure.

In view of the foregoing, it is evident that methylation of CpGdinucleotides is an important epigenetic mechanism for controllingtranscriptional activity of a cell. Generally, methylation of CpGdinucleotides correlates with transcriptional inactivity. Yet, duringnormal or degenerated differentiation processes the methylation patternof genloci may change. Accordingly, the reversal of normal methylationpatterns during tumorigenesis can lead to an abnormal repression (oractivation) of genes, for instance, tumor suppressor genes or oncogenes,respectively, and, thus, leading to tumorigenesis. Hence, the detectionof CpG methylated DNA and thus the identification of misregulatedtumor-suppressor genes and/or oncogenes is of outmost clinical interest.As mentioned above, the prior art describes different approaches for thedetection of methylated DNA which, however, suffer from certainshortcomings. For example, the methods of the prior art may not allow aneutral, genome-wide analysis of CpG methylated DNA or may not besuitable for high-through put applications or may not reliable detectCpG methylated DNA, particularly if only low amounts of DNA can be madesubject of an analysis. Thus, there is still a need for further meansand methods for detecting methylated DNA which may overcome theshortcomings and drawbacks of the prior art. Accordingly, the technicalproblem underlying the present invention is to comply with the needsdescribed above.

The solution to this technical problem is achieved by providing theembodiments characterized in the claims.

Accordingly, a first aspect of the present invention is a polynucleotidehaving a nucleotide sequence encoding a bifunctional polypeptidecomprising the DNA-binding domain of a protein belonging to the familyof Methyl-CpG binding proteins (MBDs) and an Fc portion of an antibody.Said DNA-binding domain is described herein below. It may in analternative embodiment of the present invention also be a fragmentthereof as long as said fragment is capable of binding methylated DNA,preferably CpG methylated DNA. In a preferred embodiment of the presentinvention, the nucleic acid molecule comprising a nucleotide sequenceencoding the bifunctional polypeptide of the present invention furthercomprises a nucleotide sequence encoding a linker polypeptide.Preferably, the nucleotide sequence encoding said linker polypeptide isdisposed in the polynucleotide encoding the bifunctional polypeptide ofthe present invention between the nucleotide sequence encoding the MBDand an Fc portion such that it results in a fusion between said MBD,linker polypeptide and Fc portion. A “fusion” refers to a co-linearlinkage of two or more proteins or fragments thereof via theirindividual peptide backbones through genetic expression of a nucleicacid molecule encoding those proteins. Thus, preferred fusion proteinsinclude the DNA-binding domain of an MBD or fragment thereof, whereinsaid fragment has preferably the activity of binding methylated DNA,preferably CpG methylated DNA, covalently linked to the linkerpolypeptide which is itself covalently linked to an Fc portion of anantibody as is described herein.

Said polypeptide linker is preferably a flexible linker. Preferably, itcomprises plural, hydrophilic, peptide-bonded amino acids and connectsthe C-terminal end of the DNA-binding domain of an MBD and theN-terminal end of an Fc portion. Optionally, the polypeptide of thepresent invention contains a protease cleavage site preceeding the Fcportion which allows the cut off said Fc portion if desirable. Proteasecleavage sites are, for example, a thrombin cleavage site.

Preferably, said polypeptide linker comprises a plurality of glycine,alanine, aspartate, glutamate, proline, isoleucine and/or arginineresidues. It is further preferred that said polypeptide linker comprisesa plurality of consecutive copies of an amino acid sequence. Usually,the polypeptide linker comprises 1 to 20, preferably 1 to 19, 1 to 18, 1to 17, 1 to 16 or 1 to 15 amino acids although polypeptide linkers ofmore than 20 amino acids may work as well. In a preferred embodiment ofthe invention said polypeptide linker comprises 1 to 14 amino acidresidues. In a particularly preferred embodiment of the presentinvention said polypeptide linker in the polypeptide of the inventioncomprises 14 amino acids. As demonstrated in the appended examples, saidpolypeptide linker advantageously comprises the amino acid sequence“AAADPIEGRGGGGG” which is also shown in SEQ ID NO: 2 (FIG. 1) frompositions 116 to 129.

The polypeptide of the present invention may optionally comprise a tagat its N- and/or C-Terminus. A “tag” is an amino acid sequence which ishomologous or heterologous to an amino acid sequence to which it isfused. Said tag may, inter alia, facilitate purification of a protein orfacilitate detection of said protein to which it is fused. Preferably,said tag is selected from the group consisting of a HA-tag, myc6-tag,flag-tag, strep-tag, strepII-tag, TAP-tag, HAT-tag, chitin bindingdomain (CBD), maltose-binding protein, immunoglobulin A (IgA),His-6-tag, glutathione-S-transferase (GST) tag, intein and streptavidiebinding protein (SBP) tag.

CpG islands frequently contain gene promoters and transcription startsites and are usually unmethylated in normal cells. Methylation ofCpG-islands is associated with transcriptional repression. In cancer,the methylation of CpG-island promoters leads to the abnormal silencingof tumor-suppressor genes, contributing to the pathogenesis of thedisease. So far, the investigation of aberrant CpG-island methylation inhuman cancer has primarily taken a candidate gene approach which,however, suffers from several shortcomings. These are, for example,incomplete coverage of genloci involved in tumorigenesis which may besubject of methylation, either hyper- or hypomethylation or incompleteanalysis of genloci due to limited means and methods when using, forexample, Restriction Landmark Genomic Scanning (RLGS). To allow anunbiased, genome wide detection of CpG-methylated DNA, the presentinvention provides means and methods that allow the separation anddetection of CpG-methylation, without applying, for example,methylation-sensitive restriction endonucleases or bisulfite-treatment.These means and methods are, inter alia, based on a methyl-CpG-binding,antibody-like protein that efficiently binds CpG-methylated DNA. Asdescribed herein, the methyl-CpG-binding, antibody-like proteincomprises a DNA-binding domain of a protein belonging to the family ofMethyl-CpG binding proteins (MBDs) and the constant portion of anantibody.

It was surprisingly found that a recombinant methyl-CpG-binding,antibody-like protein can preferably bind CpG methylated DNA in anantibody-like manner. That means, the methyl-CpG-binding, antibody-likeprotein of the present invention has a high affinity and high avidity toits “antigen” which is preferably DNA that is preferably methylated atCpG dinucleotides. Without being bound by theory the high affinity andavidity of the polypeptide of the present invention for its “antigen” iscaused by the unique structure of said methyl-CpG-binding, antibody-likeprotein. The unique structure of the polypeptide of the presentinvention is assumed to be achieved by the presence of the constantregion of an antibody and, thus, renders said polypeptide to bepreferably a bifunctional molecule. The constant regions are believed toform disulfide-bonds between immunoglobulin heavy chains of the constantregions of each of two polypeptide molecules of the present invention.Accordingly, preferably an antibody-like structure is formed closelyresembling the structure of an antibody.

Again, without being bound by theory it is assumed that this structurelends, for example, stability on the polypeptide of the presentinvention. This is because, it is described in the art that proteinsfused to a constant region of an antibody may confer a higher stabilityand half-life of the said protein. In addition, it is believed that theantibody-like structure caused by the intermolecular interaction of theconstant regions brings the methyl-DNA-binding domain of one polypeptideof the present invention in close proximity to the methyl-DNA-bindingdomain of another polypeptide of the present invention. This allowsbivalent interactions between the methyl-DNA-binding proteins andmethylated DNA. Accordingly, the polypeptide of the present invention ispreferably capable of binding to its antigen via two methyl DNA-bindingdomains which are part of the polypeptide of the present invention. Thehigh affinity binding of the polypeptide of the present invention is,inter alia, also achieved by using preferably methyl-DNA-binding domainsof proteins instead of the full-length methyl-DNA-binding proteincontaining domains for the interaction with other proteins that may,however, disturb or interfere the unique applicability as describedherein which are known to specifically bind to methylated DNA,preferably, CpG methylated DNA, rather than to unmethylated DNA. The useof the methyl-DNA-binding domain, moreover, guarantees that indeedmethylated DNA is bound since the detection is direct and not indirect.Most prior art methods can only indirectly detect methylated DNA by PCR.

These properties award the polypeptide of the present invention to be areliable and easy applicable diagnostic tool for, inter alia, isolating,purifying enriching and/or detecting methylated DNA even if said DNA isonly present in very small amounts, e.g., about more than 10 ng, lessthan 10 ng, less than 7.5 ng, less than 5 ng, less than 2.5 ng or about1 ng as described herein. Accordingly, due to its antibody-likestructure the polypeptide of the present invention is a robust moleculerendering it to be applicable, for instance, for various applicationsincluding multi-step procedures in a single tube assay. For example,specific separation and detection of CpG-methylated DNA was demonstratedusing reverse South-Western blot analysis and methyl-CpGimmunoprecipitation (MCIp). The latter technique, combined withreal-time PCR, e.g. LightCycler PCR, allows the sensitive detection ofCpG-island methylation of candidate CpG-island promoters from as littleas, e.g., 1 ng total genomic DNA. MCIp-generated genomic DNA-fragmentscan be easily amplified, labelled and used for CpG-island microarrayhybridisation. Using the techniques described herein, it is possible togenerate genome-wide profiles of aberrant CpG-island methylation inhuman cancer and, for example, to identify (a) tumor-suppressor gene(s)or further suppressor gene activities.

Before the present invention is described in detail, it is to beunderstood that this invention is not limited to the particularmethodology, protocols, bacteria, vectors, and reagents etc. describedherein as these may vary. It is also to be understood that theterminology used herein is for the purpose of describing particularembodiments only, and is not intended to limit the scope of the presentinvention which will be limited only by the appended claims. Unlessdefined otherwise, all technical and scientific terms used herein havethe same meanings as commonly understood by one of ordinary skill in theart.

Preferably, the terms used herein are defined as described in “Amultilingual glossary of biotechnological terms: (IUPACRecommendations)”, Leuenberger, H. G. W, Nagel, B. and Kölbl, H. eds.(1995), Helvetica Chimica Acta, CH-4010 Basel, Switzerland). Throughoutthis specification and the claims which follow, unless the contextrequires otherwise, the word “comprise”, and variations such as“comprises” and “comprising”, will be understood to imply the inclusionof a stated integer or step or group of integers or steps but not theexclusion of any other integer or step or group of integer or step. Itmust be noted that as used herein and in the appended claims, thesingular forms “a”, “an”, and “the”, include plural referents unless thecontext clearly indicates otherwise. Thus, for example, reference to “areagent” includes one or more of such different reagents, and referenceto “the method” includes reference to equivalent steps and methods knownto those of ordinary skill in the art that could be modified orsubstituted for the methods described herein.

The term “nucleic acid molecule” when used herein encompasses anynucleic acid molecule having a nucleotide sequence of bases comprisingpurine- and pyrimidine bases which are comprised by said nucleic acidmolecule, whereby said bases represent the primary structure of anucleic acid molecule. Nucleic acid sequences include DNA, cDNA, genomicDNA, RNA, synthetic forms, for example, PNA, and mixed polymers, bothsense and antisense strands, or may contain non-natural or derivatizednucleotide bases, as will be readily appreciated by those skilled in theart.

The polynucleotide of the present invention is preferably composed ofany polyribonucleotide or polydeoxribonucleotide, which may beunmodified RNA or DNA or modified RNA or DNA. For example, thepolynucleotide can be composed of single- and double-stranded DNA, DNAthat is a mixture of single- and double-stranded regions, single- anddouble-stranded RNA, and RNA that is mixture of single- anddouble-stranded regions, hybrid molecules comprising DNA and RNA thatmay be single-stranded or, more typically, double-stranded or a mixtureof single- and double-stranded regions. In addition, the polynucleotidecan be composed of triple-stranded regions comprising RNA or DNA or bothRNA and DNA. The polynucleotide may also contain one or more modifiedbases or DNA or RNA backbones modified for stability or for otherreasons. “Modified” bases include, for example, tritylated bases andunusual bases such as inosine. A variety of modifications can be made toDNA and RNA; thus, the term “nucleic acid molecules” embraceschemically, enzymatically, or metabolically modified forms.

The term “polypeptide” when used herein means a peptide, a protein, or apolypeptide which are used interchangeable and which encompasses aminoacid chains of a given length, wherein the amino acid residues arelinked by covalent peptide bonds. However, peptidomimetics of suchproteins/polypeptides wherein amino acid(s) and/or peptide bond(s) havebeen replaced by functional analogs are also encompassed by theinvention as well as other than the 20 gene-encoded amino acids, such asselenocysteine. Peptides, oligopeptides and proteins may be termedpolypeptides. As mentioned the terms polypeptide and protein are oftenused interchangeably herein. The term polypeptide also refers to, anddoes not exclude, modifications of the polypeptide. Modificationsinclude glycosylation, acetylation, acylation, phosphorylation,ADP-ribosylation, amidation, covalent attachment of flavin, covalentattachment of a heme moiety, covalent attachment of a nucleotide ornucleotide derivative, covalent attachment of a lipid or lipidderivative, covalent attachment of phosphotidylinositol, cross-linking,cyclization, disulfide bond formation, demethylation, formation ofcovalent cross-links, formation of cysteine, formation of pyroglutamate,formulation, gamma-carboxylation, glycosylation, GPI anchor formation,hydroxylation, iodination, methylation, myristoylation, oxidation,pegylation, proteolytic processing, phosphorylation, prenylation,racemization, selenoylation, sulfation, transfer-RNA mediated additionof amino acids to proteins such as arginylation, and ubiquitination;see, for instance, PROTEINS—STRUCTURE AND MOLECULAR PROPERTIES, 2nd Ed.,T. E. Creighton, W. H. Freeman and Company, New York (1993);POST-TRANSLATIONAL COVALENT MODIFICATION OF PROTEINS, B. C. Johnson,Ed., Academic Press, New York (1983), pgs. 1-12; Seifter, Meth. Enzymol.182 (1990); 626-646, Rattan, Ann. NY Acad. Sci. 663 (1992); 48-62.

The polypeptide of the present invention has preferably the amino acidsequence encoded by a nucleic acid molecule of the present invention asdescribed herein or is obtainable by a process for producing saidpolypeptide or by a process for producing cells capable of expressingsaid polypeptide which is described herein.

Preferably, in the context of the present invention the polypeptide is abifunctional polypeptide. A “bifunctional polypeptide” means that thepolypeptide of the present invention has, in addition to binding tomethylated DNA, preferably to CpG methylated DNA, due to an Fc portionof an antibody which is part of the polypeptide of the presentinvention, further capabilities. For example, said Fc portion preferablyoffers the possibility to conjugate, link or covalently couple (a)compound(s) or moieties to said Fc portion. As used herein, the term“covalently coupled” means that the specified compounds or moieties areeither directly covalently bonded to one another, or else are indirectlycovalently joined to one another through an intervening moiety ormoieties, such as a bridge, spacer, or linkage moiety or moieties.

Such (a) compound(s) may be a detectable substance. Examples ofdetectable substances include various enzymes, prosthetic groups,fluorescent materials, luminescent materials, bioluminescent materials,radioactive materials, positron emitting metals using various positronemission tomographies, and nonradioactive paramagnetic metal ions. Thedetectable substance may be coupled or conjugated either directly to anFc portion of an antibody (or fragment thereof) or indirectly, throughan intermediate (such as, for example, a linker known in the art) usingtechniques known in the art. See, for example, U.S. Pat. No. 4,741,900for metal ions which can be conjugated to an Fc portion of antibodiesfor use as diagnostics according to the present invention. Examples ofsuitable enzymes include horseradish peroxidase, alkaline phosphatase,beta-galactosidase, or acetylcholinesterase; examples of suitableprosthetic group complexes include streptavidin/biotin andavidin/biotin; examples of suitable fluorescent materials includeumbelliferone, fluorescein, fluorescein isothiocyanate, rhodamine,dichlorotriazinylamine fluorescein, dansyl chloride or phycoerythrin; anexample of a luminescent material includes luminol; examples ofbioluminescent materials include luciferase, luciferin, and aequorin;and examples of suitable radioactive material include ¹²⁵I, ¹³¹I, or⁹⁹Tc.

Further, said Fc portion may be conjugated to a therapeutic moiety suchas a cytotoxin, e.g., a cytostatic or cytocidal agent, a therapeuticagent or a radioactive metal ion, e.g., alpha-emitters such as, forexample, ²¹³Bi. A cytotoxin or cytotoxic agent includes any agent thatis detrimental to cells. Examples include paclitaxol, cytochalasin B,gramicidin D, ethidium bromide, emetine, mitomycin, etoposide,tenoposide, vincristine, vinblastine, colchicin, doxorubicin,daunorubicin, dihydroxy anthracin dione, mitoxantrone, mithramycin,actinomycin D, 1-dehydrotestosterone, glucocorticoids, procaine,tetracaine, lidocaine, propranolol, and puromycin and analogs orhomologues thereof. Therapeutic agents include, but are not limited to,antimetabolites (e.g., methotrexate, 6-mereaptopurine, 6-thioguanine,cytarabine, 5-fluorouracil decarbazine), alkylating agents (e.g.,mechlorethamine, thioepa chlormbucil, melphalan, carmustine (BSNU) andlomustine (CCNU), cyclothosphamide, busulfan, dibromomannitol,streptozotocin, mitomycin C, and cis-dichlorodiamine platinum (11) (DDP)cisplatin), anthracyclines (e.g., daunorubicin (formerly daunomycin) anddoxorubicin), antibiotics (e.g., dactinomycin (formerly actinomycin),bleomycin, mithramycin, and anthramycin (AMC)), and anti-mitotic agents(e.g., vincristine and vinblastine).

Furthermore, the Fc portion of the polypeptide of the present inventionmay be coupled or conjugated to a protein or polypeptide possessing adesired biological activity. Such proteins may include, for example, atoxin such as abrin, ricin A, pseudomonas exotoxin, or diphtheria toxin;a protein such as tumor necrosis factor, α-interferon, β-interferon,nerve growth factor, platelet derived growth factor, tissue plasminogenactivator, an apoptotic agent.

The Fc portion also allows attachment of the polypeptide of the presentinvention to solid supports, which are particularly useful forimmunoassays or purification of the target artigen as described herein.Such solid supports include, but are not limited to, glass, cellulose,polyacrylamide, nylon, polycabonate, polystyrene, polyvinyl chloride orpolypropylene or the like.

Techniques for conjugating coupling or linked compounds to the Fcportion are well known, see, e.g., Arnon et al., “Monoclonal AntibodiesFor Immunotargeting Of Drugs In Cancer Therapy”, in MonoclonalAntibodies And Cancer Therapy, Reisfeld et al. (eds.), pp. 243-56 (AlanR. Liss, Inc. 1985); Hellstrom et al., “Antibodies For Drug Delivery”,in Controlled Drug Delivery (2nd Ed.), Robinson et al. (eds.), pp.623-53 (Marcel Dekker, Inc. 1987); Thorpe, “Antibody Carriers OfCytotoxic Agents In Cancer Therapy: A Review”, in Monoclonal Antibodies‘84: Biological And Clinical Applications, Pinchera et al. (eds.), pp.475-506 (1985); “Analysis, Results, And Future Prospective Of TheTherapeutic Use Of Radiolabeled Antibody In Cancer Therapy’”, inMonoelonal Antibodies For Cancer Detection And Therapy, Baldwin et al.(eds.), pp. 303-16 (Academic Press 1985), and Thorpe, Immunol. Rev.,119-158.

The term “DNA-binding domain of a protein belonging to the family ofMethyl-CpG binding proteins (MBDs)” encompasses a polypeptide which haspreferably the structural and/or functional characteristics of themethyl-DNA-binding domain of a protein of the MBD family which comprisesthe proteins MeCP2, MBD1, MBD2, MBD3 and MBD4. The methyl-DNA-bindingactivity can be tested by methods known in the art. Preferably, the term“methylated DNA” encompasses methylated DNA, more preferably, CpGmethylated DNA including hemi-methylated or DNA methylated at bothstrands or single-stranded, methylated DNA. The most important exampleto date is methylated cytosine that occurs mostly in the context of thedinucleotide CpG, but also in the context of CpNpG- and CpNpN-sequences.In principle, other naturally occurring nucleotides may also bemethylated.

It is preferred that the polypeptide of the present invention bindsmethylated DNA either as a monomer or dimer or multivalent molecule asdescribed herein. It is preferably capable of binding to highlymethylated DNA or low methylated DNA. Preferably, it can bind singlemethylated CpG pairs. MeCP2, MBD1, MBD2, MBD3 and MBD4 constitute afamily of vertebrate proteins that share the methyl-CpG-binding domain.The MBD protein family comprises two subgroups based upon sequences ofthe known MBDs. The methyl-DNA-binding domain of MBD4 is most similar tothat of MeCP2 in primary sequence, while the methyl-DNA-binding domainof MBD1, MBD2 and MBD3 are more similar to each other than to those ofeither MBD4 or MeCP2. However, the methyl-DNA-binding domains withineach protein appear to be related evolutionarily based on the presenceof an intron located at a conserved position within all five genes ofMeCP2, MBD1, MBD2, MBD3 and MBD4. Yet, the sequence similarity betweenthe members of the MBD family is largely limited to theirmethyl-DNA-binding domain, although MBD2 and MBD3 are similar and shareabout 70% of overall identity over most of their length. The greatestdivergence occurs at the C-terminus, where MBD3 has 12 consecutiveglutamic acid residues.

An MBD or fragment thereof preferably a methyl-DNA-binding domain orfragment thereof useful in accordance with the present invention can,for example, be identified by using sequence comparisons and/oralignments by employing means and methods known in the art, preferablythose described herein and comparing and/or aligning (a) known MBD(s)to/with a sequence suspected to be an MBD.

For example, when a position in both of the two compared sequences isoccupied by the same base or amino acid monomer subunit (for instance,if a position in each of the two DNA molecules is occupied by adenine,or a position in each of two polypeptides is occupied by a lysine), thenthe respective molecules are identical at that position. The percentageidentity between two sequences is a function of the number of matchingor identical positions shared by the two sequences divided by the numberof positions compared×100. For instance, if 6 of 10 of the positions intwo sequences are matched or are identical, then the two sequences are60% identical. By way of example, the DNA sequences CTGACT and CAGGTTshare 50% homology (3 of the 6 total positions are matched). Generally,a comparison is made when two sequences are aligned to give maximumhomology and/or identity. Such alignment can be provided using, forinstance, the method of Needleman, J. Mol Biol. 48 (1970): 443-453,implemented conveniently by computer programs such as the Align program(DNAstar, Inc.). Homologous sequences share identical or similar aminoacid residues, where similar residues are conservative substitutionsfor, or “allowed point mutations” of, corresponding amino acid residuesin an aligned reference sequence. In this regard, a “conservativesubstitution” of a residue in a reference sequence are thosesubstitutions that are physically or functionally similar to thecorresponding reference residues, e.g., that have a similar size, shape,electric charge, chemical properties, including the ability to formcovalent or hydrogen bonds, or the like. Particularly preferredconservative substitutions are those fulfilling the criteria defined foran “accepted point mutation” in Dayhoff et al., 5: Atlas of ProteinSequence and Structure, 5: Suppl. 3, chapter 22: 354-352, Nat. Biomed.Res. Foundation, Washington, D.C. (1978).

Preferably, a methyl-DNA-binding domain or fragment thereof of thepolypeptide of the present invention has the structural and/orfunctional characteristics as described herein. Preferably, a fragmentof a methyl-DNA-binding protein described herein is able to bindmethylated DNA, preferably CpG methylated DNA.

The methyl-DNA-binding domain or fragment thereof of the polypeptide ofthe present invention is preferably of insect origin, nematode origin,fish origin, amphibian origin, more preferably of vertebrate origin,even more preferably of mammal origin, most preferably of mouse andparticularly preferred of human origin.

Preferably, the methyl-DNA-binding domain or fragment thereof of thepolypeptide of the present invention possesses a uniquealpha-helix/beta-strand sandwich structure with characteristic loops asis shown in FIG. 1 of Ballester and Wolffe, Eur. J. Biochem. 268 (2001),1-6 and is able to bind methylated DNA.

More preferably, the MBD or fragment thereof of the polypeptide of thepresent invention comprises at least 50, more preferably at least 60,even more preferably at least 70 or at least 80 amino acid residues ofthe MBDs shown in FIG. 1 of Ballester and Wolffe (2001), loc. cit. andis able to bind methylated DNA.

Even more preferably, the methyl-DNA-binding domain or fragment thereofof the polypeptide of the present invention shares preferably 50%, 60%,70%, 80% or 90%, more preferably 95% or 97%, even more preferably 98%and most preferably 99% identity on amino acid level to the MBDs shownin FIG. 1 of Ballester and Wolffe (2001), loc. cit. and is able to bindmethylated DNA. Means and methods for determining the identity ofsequences, for example, amino acid sequences is described elsewhereherein.

Most preferably, the methyl-DNA-binding domain or fragment thereof ofthe polypeptide of the present invention comprises themethyl-DNA-binding domain of the MBD proteins shown in FIG. 1 ofBallester and Wolffe (2001), loc. cit. or the methyl-DNA-binding domainof the MBD proteins described in Hendrich and Tweedy, Trends Genet. 19(2003), 269-77 and is able to bind methylated DNA.

Of course, in accordance with the present invention, the polypeptide ofthe present invention is preferably bifunctional and harbours preferablytwo methyl-DNA-binding domains as described above, wherein preferablyboth methyl-DNA-binding domains are able to bind single methylated CpGpairs.

In a particular preferred embodiment of the invention, themethyl-DNA-binding domain of the polypeptide of the present invention isthat of human MBD2. In a more particular preferred embodiment, themethyl-DNA-binding domain is that of human MBD2 comprising amino acids144 to 230 of the amino acid sequence having Genbank accession numberNM_(—)003927. In a most particular preferred embodiment, themethyl-DNA-binding domain of the polypeptide of the present inventioncomprises the amino acid sequence from position 29 to 115 of the aminoacid sequence shown in SEQ ID NO:2 (FIG. 1).

An “Fc portion” of an antibody which is a component of the polypeptideof the present invention comprises preferably at least a portion of theconstant region of an immunoglobulin heavy chain molecule. The Fc regionis preferably limited to the constant domain hinge region and the C_(H)2and C_(H)3 domains. The Fc region in the polypeptide of the presentinvention can also be limited to a portion of the hinge region, theportion being capable of forming intermolecular disulfide bridges, andthe C_(H)2 and C_(H)3 domains, or functional equivalents thereof.

Alternatively, it is also preferred that the Fc portion comprises atleast so many C_(H) regions which are required such that the polypeptideof the present invention has still the properties of the polypeptidedescribed hereinabove, in particular the properties of the polypeptideused in the appended Examples.

In another alternative, it is also preferred that said constant regionmay contain one or more amino acid substitutions when compared toconstant regions known in the art. Preferably it contains 1 to 100, 1 to90, 1 to 80, 1 to 70, 1 to 60, 1 to 50, 1 to 40, 1 to 30 or 1 to 20,more preferably 1 to 10, even more preferably 1 to 9, 1 to 8, 1 to 7 or1 to 6 and most preferably 1 to 5, 1 to 4, 1 to 3 or 2 or 1substitution(s). The comparison is preferably done as is known in theart or, more preferably, as described elsewhere herein.

Alternatively, said constant region comprises preferably at least theC_(H)1 region, more preferably the C_(H)1 and C_(H)2 regions and mostpreferably the C_(H)1, C_(H)2 and C_(H)3 region. As is known in the art,the constant region of an antibody contains two immunoglobulin heavychains which harbour three characteristic immunoglobulin domainscomposed of about 110 amino acids, wherein the two immunoglobulin heavychains are covalently linked via disulfide bonds. Without being bound bytheory, it is believed that the nascent polypeptide of the presentinvention comprising an methyl-DNA-binding domain and an Fc portion ofan antibody is folded within a host cell such that preferably twopolypeptides are joined at their Fc portion in a manner similar or,preferably, identical to the constant region of an antibody, resultingin a bifunctional polypeptide as described herein.

It is also envisaged that the constant region could preferably be ofchicken or duck origin. Yet, preferably, the constant region is of theIgM, IgA, IgD or IgE isotype and more preferably it is of the IgGisotype, most preferably of the IgG1 isotype. Preferably, theaforementioned isotypes are of vertebrate origin, more preferably ofmammal origin, even more preferably of mouse, rat, goat, horse, donkey,camel or chimpanzee origin and most preferably of human origin.Preferably, said IgG isotype is of class IgG1, IgG2, IgG3, IgG4 and saidIgA isotype is of class IgA1, IgA2.

As described herein, the present invention provides preferably forbifunctional polypeptides. Yet, also multimeric bifunctionalpolypeptides comprising one or more of the bifunctional polypeptide ofthe present invention are envisaged. Such multimers may be generated byusing those Fc regions, or portions thereof, of Ig molecules which areusually multivalent such as IgM pentamers or IgA dimers. It isunderstood that a J chain polypeptide may be needed to form andstabilize IgM pentamers and IgA dimers.

In a more preferred embodiment, the nucleic acid molecule comprising anucleotide sequence of the present invention described hereinabove isselected from the group consisting of:

-   (a) a nucleic acid sequence having the nucleotide sequence shown in    SEQ ID NO: 1 (FIG. 1);-   (b) a nucleic acid sequence having a nucleotide sequence encoding a    polypeptide having the amino acid sequence shown in SEQ ID: NO 2    (FIG. 1);-   (c) a nucleic acid sequence having a nucleotide sequence encoding a    fragment of a polypeptide having the amino acid sequence shown in    SEQ ID: NO 2 (FIG. 1), wherein said fragment comprises at least    amino acids 130 to 361 of said polypeptide and which is capable of    binding methylated DNA;-   (d) a nucleic acid sequence having a nucleotide sequence encoding a    variant of a polypeptide encoded by a polynucleotide of any one    of (a) to (c), wherein in said variant one or more amino acid    residues are substituted compared to said polypeptide, and said    variant is capable of binding methylated DNA;-   (e) a nucleic acid sequence having a nucleotide sequence which    hybridizes with a nucleic acid sequence of any one of (a) to (d) and    which is at least 65% identical to the nucleotide sequence of the    nucleic acid molecule of (a) and which encodes a polypeptide being    capable of binding methylated DNA;-   (f) a nucleic acid molecule encoding a polypeptide which is at least    65% identified to a polypeptide encoded by a nucleic acid molecule    of (b) and which is capable of binding methylated DNA; and-   (g) a nucleic acid sequence having a nucleotide sequence being    degenerate to the nucleotide sequence of the polynucleotide of any    one of (a) to (f);    or the complementary strand of such a polynucleotide.

As described above, the fragment of the polypeptide of the presentinvention having the amino acid sequence shown in SEQ ID: NO 2 (FIG. 1)comprises at least amino acids 130 to 361 of the amino acid sequenceshown in SEQ ID: NO 2 (FIG. 1). That means that said fragment maycomprise in addition to amino acids 130 to 361 which represent the Fcportion, one or more amino acids such that said fragment is capable ofbinding methylated DNA, preferably, CpG methylated DNA, rather thanunmethylated DNA. Accordingly, it is envisaged that said fragmentcomprises more preferably, at least amino acids 116 to 361 of the aminoacid sequence shown in SEQ ID: NO 2 (FIG. 1). Even more preferably, saidfragment may comprise at least amino acids 29 to 115 and 130 to 361 ofthe amino acid sequence shown in SEQ ID: NO 2 (FIG. 1). In a mostpreferred embodiment, said fragment may comprise at least amino acids 29to 361. It is generally preferred that the fragments of the polypeptideof the present invention are able to bind to methylated DNA, preferablyto CpG methylated DNA, rather than unmethylated DNA. This ability can betested by methods known in the art or preferably by those methodsdescribed in the appended Examples.

A “variant” of the polypeptide of the present invention encompasses apolypeptide wherein one or more amino acid residues are substituted,preferably conservatively substituted compared to said polypeptide andwherein said variant is preferably able to bind to methylated DNA,preferably CpG methylated DNA. Such variants include deletions,insertions, inversions, repeats, and substitutions selected according togeneral rules known in the art so as have no effect on the activity ofthe polypeptide of the present invention. For example, guidanceconcerning how to make phenotypically silent amino acid substitutions isprovided in Bowie, Science 247: (1990) 1306-1310, wherein the authorsindicate that there are two main strategies for studying the toleranceof an amino acid sequence to change.

The first strategy exploits the tolerance of amino acid substitutions bynatural selection during the process of evolution. By comparing aminoacid sequences in different species, conserved amino acids can beidentified. These conserved amino acids are likely important for proteinfunction. In contrast, the amino acid positions where substitutions havebeen tolerated by natural selection indicates that these positions arenot critical for protein function. Thus, positions tolerating amino acidsubstitution could be modified while still maintaining biologicalactivity of the protein.

The second strategy uses genetic engineering to introduce amino acidchanges at specific positions of a cloned gene to identify regionscritical for protein function. For example, site directed mutagenesis oralanine-scanning mutagenesis (introduction of single alanine mutationsat every residue in the molecule) can be used. (Cunningham and Wells,Science 244: (1989) 1081-1085.) The resulting mutant molecules can thenbe tested for biological activity.

As the authors state, these two strategies have revealed that proteinsare surprisingly tolerant of amino acid substitutions. The authorsfurther indicate which amino acid changes are likely to be permissive atcertain amino acid positions in the protein. For example, most buried(within the tertiary structure of the protein) amino acid residuesrequire nonpolar side chains, whereas few features of surface sidechains are generally conserved.

The invention encompasses polypeptides having a lower degree of identitybut having sufficient similarity so as to perform one or more of thefunctions performed by the polypeptide of the present invention.Similarity is determined by conserved amino acid substitution. Suchsubstitutions are those that substitute a given amino acid in apolypeptide by another amino acid of like characteristics (e.g.,chemical properties). According to Cunningham et al. above, suchconservative substitutions are likely to be phenotypically silent.Additional guidance concerning which amino acid changes are likely to bephenotypically silent are found in Bowie, Science 247: (1990) 1306-1310.

Tolerated conservative amino acid substitutions of the present inventioninvolve replacement of the aliphatic or hydrophobic amino acids Ala,Val, Leu and Ile; replacement of the hydroxyl residues Ser and Thr;replacement of the acidic residues Asp and Glu; replacement of the amideresidues Asn and Gln, replacement of the basic residues Lys, Arg, andHis; replacement of the aromatic residues Phe, Tyr, and Trp, andreplacement of the small-sized amino acids Ala, Ser, Thr, Met, and Gly.

In addition, the present invention also encompasses the conservativesubstitutions provided in the Table below.

TABLE IV For Amino Aeid Code Replace with any of: Alanine A D-Ala, Gly,beta-Ala, L-Cys, D-C_(y)s Arginine R D-Arg, Lys, D-Lys, homo-Arg,D-homo-Arg, Met, Ile, D-Met, D-Ile, Orn, D-Orn Asparagine N D-Asn, Asp,D-Asp, Glu, D-Glu, Gln, D-Gln Aspartic Acid D D-Asp, D-Asn, Asn, Glu,D-Glu, Gln, D-Gln Cysteine C D-Cys, S-Me-Cys, Met, D-Met, Thr, D-ThrGlutamine Q D-Gln, Asn, D-Asn, Glu, D-Glu, Asp, D-As Glutamic Acid ED-Glu, D-Asp, Asp, Asn, D-Asn, Gln, D-Gln Glycine G Ala, D-Ala, Pro,D-Pro, β-Ala, Acp Isoleucine D-Ile, Val, D-Val, Leu, D-Leu, Met, D-MetLeucine L D-Leu, Val, D-Val, Met, D-Met Lysine K D-Lys, Arg, D-Arg,homo-Arg, D-homo-Arg, Met, D-Met, Ile, D-Ile, Orn, D-Orn Methionine MD-Met, S-Me-Cys, Ile, D-Ile, Leu, D-Leu, Val, D-Val Phenylalanine FD-Phe, Tyr, D-Thr, L-Dopa, His, D-His, Trp, D-Trp, Trans-3,4, or5-phenylproline, cis-3,4, or 5-phenylproline Proline P D-Pro,L-1-thioazolidine-4-carboxylic acid, D- or L-1-oxazolidine-4-carboxylicacid Serine S D-Ser, Thr, D-Thr, allo-Thr, Met, D-Met, Met(O), D-Met(O),L-Cys, D-Cys Threonine T D-Thr, Ser, D-Ser, allo-Thr, Met, D-Met,Met(O), D-Met(O), Val, D-Val Tyrosine Y D-Tyr, Phe, D-Phe, L-Dopa, His,D-His Valine V D-Val, Leu, D-Leu, Ile, D-Ile, Met, D-Met

Aside from the uses described above, such amino acid substitutions mayalso increase protein or peptide stability. The invention encompassesamino acid substitutions that contain, for example, one or morenon-peptide bonds (which replace the peptide bonds) in the protein orpeptide sequence. Also included are substitutions that include aminoacid residues other than naturally occurring L-amino acids, e.g.,D-amino acids or non-naturally occurring or synthetic amino acids, e.g.,R or y amino acids.

Both identity and similarity can be readily calculated by reference tothe following publications: Computational Molecular Biology, Lesk, A.M., ed., Oxford University Press, New York, 1988; Biocomputing:Infoliuaties and Genome Projects, Smith, D M., ed., Academic Press, NewYork, 1993; Informafies Computer Analysis of Sequence Data, Part 1,Griffin, A. M., and Griffin, H. G., eds., Humana Press, New Jersey,1994; Sequence Analysis in Molecular Biology, von Heinje, G., AcademiePress, 1987; and Sequence Analysis Primer, Gribskov, M. and Devereux,eds., M Stockton Press, New York, 1991.

As described above, the present invention preferably also relates tonucleic acid sequences which hybridize to the nucleic acid sequenceshown in SEQ ID NO: 1 fragments or variants thereof as described herein(FIG. 1) and which are at least 65% identical to the nucleic acidsequence shown in SEQ ID NO: 1 (FIG. 1) and which preferably encode apolypeptide being capable of binding methylated DNA, preferably CpGmethylated DNA, rather than unmethylated DNA. As also described, thepresent invention preferably relates to nucleic acid sequences encodinga polypeptide which is at least 65%, more preferably 70%, 75%, 80%, 85%,90%, more preferably 99% identified to the polypeptide shown in SEQ IDNO:2. The term “hybridizes” as used in accordance with the presentinvention preferably relates to hybridizations under stringentconditions. The term “hybridizing sequences” preferably refers tosequences which display a sequence identity of at least 65%, even morepreferably at least 70%, particularly preferred at least 80%, moreparticularly preferred at least 90%, even more particularly preferred atleast 95% and most preferably at least 97, 98% or 99% identity with anucleic acid sequence as described above encoding a polypeptide which isable to bind to methylated DNA, preferably CpG methylated DNA, ratherthan unmethylated DNA.

Said hybridization conditions may be established according toconventional protocols described, for example, in Sambrook, Russell“Molecular Cloning, A Laboratory Manual”, Cold Spring Harbor Laboratory,N.Y. (2001); Ausubel, “Current Protocols in Molecular Biology”, GreenPublishing Associates and Wiley Interscience, N.Y. (1989), or Higginsand Hames (Eds.) “Nucleic acid hybridization, a practical approach” IRLPress Oxford, Washington D.C., (1985). The setting of conditions is wellwithin the skill of the artisan and can be determined according toprotocols described in the art. Thus, the detection of only specificallyhybridizing sequences will usually require stringent hybridization andwashing conditions such as 0.1×SSC, 0.1% SDS at 65° C. Non-stringenthybridization conditions for the detection of homologous or not exactlycomplementary sequences may be set at 6×SSC, 1% SDS at 65° C. As is wellknown, the length of the probe and the composition of the nucleic acidto be determined constitute further parameters of the hybridizationconditions. Note that variations in the above conditions may beaccomplished through the inclusion and/or substitution of alternateblocking reagents used to suppress background in hybridizationexperiments. Typical blocking reagents include Denhardt's reagent,BLOTTO, heparin, denatured salmon sperm DNA, and commercially availableproprietary formulations. The inclusion of specific blocking reagentsmay require modification of the hybridization conditions describedabove, due to problems with compatibility. Hybridizing nucleic acidmolecules also comprise fragments of the above described molecules. Suchfragments may represent nucleic acid sequences as described herein.Furthermore, nucleic acid molecules which hybridize with any of theaforementioned nucleic acid molecules also include complementaryfragments, derivatives and allelic variants of these molecules.Additionally, a hybridization complex refers to a complex between twonucleic acid sequences by virtue of the formation of hydrogen bondsbetween complementary G and C bases and between complementary A and Tbases; these hydrogen bonds may be further stabilized by base stackinginteractions. The two complementary nucleic acid sequences hydrogen bondin an antiparallel configuration. A hybridization complex may be formedin solution (e.g., Cot or Rot analysis) or between one nucleic acidsequence present in solution and another nucleic acid sequenceimmobilized on a solid support (e.g., membranes, filters, chips, pins orglass slides to which, e.g., cells have been fixed). The termscomplementary or complementarity refer to the natural binding ofpolynucleotides under permissive salt and temperature conditions bybase-pairing. For example, the sequence “A-G-T” binds to thecomplementary sequence “T-C-A”. Complementarity between twosingle-stranded molecules may be “partial”, in which only some of thenucleic acids bind, or it may be complete when total complementarityexists between single-stranded molecules. The degree of complementartitybetween nucleic acid strands has significant effects on the efficiencyand strength of hybridization between nucleic acid strands. This is ofparticular importance in amplification reactions, which depend uponbinding between nucleic acids strands.

In accordance with the present invention, the term “identical” or“percent identity” in the context of two or more nucleic acid or aminoacid sequences, refers to two or more sequences or subsequences that arethe same, or that have a specified percentage of amino acid residues ornucleotides that are the same (e.g., at least 65% identity, preferably,at least 70-95% identity, more preferably at least 95%, 96%, 97%, 98% or99% identity), when compared and aligned for maximum correspondence overa window of comparison, or over a designated region as measured using asequence comparison algorithm as known in the art, or by manualalignment and visual inspection. Sequences having, for example, 65% to95% or greater sequence identity are considered to be substantiallyidentical. Such a definition also applies to the complement of a testsequence. Preferably the described identity exists over a region that isat least about 232 amino acids or 696 nucleotides in length. Thosehaving skill in the art will know how to determine percent identitybetween/among sequences using, for example, algorithms such as thosebased on CLUSTALW computer program (Thompson Nucl. Acids Res. 2 (1994),4673-4680) or FASTDB (Brutlag Comp. App. Biosci. 6 (1990), 237-245), asknown in the art.

Although the FASTDB algorithm typically does not consider internalnon-matching deletions or additions in sequences, i.e., gaps, in itscalculation, this can be corrected manually to avoid an overestimationof the % identity. CLUSTALW, however, does take sequence gaps intoaccount in its identity calculations. Also available to those havingskill in this art are the BLAST and BLAST 2.0 algorithms (Altschul Nucl.Acids Res. 25 (1977), 3389-3402). The BLASTN program for nucleic acidsequences uses as defaults a word length (W) of 11, an expectation (E)of 10, M=5, N=4, and a comparison of both strands. For amino acidsequences, the BLASTP program uses as defaults a wordlength (W) of 3,and an expectation (E) of 10. The BLOSUM62 scoring matrix (HenikoffProc. Natl. Acad. Sci., USA, 89, (1989), 10915) uses alignments (B) of50, expectation (E) of 10, M=5, N=4, and a comparison of both strands.For example, BLAST2.0, which stands for Basic Local Alignment SearchTool (Altschul, Nucl. Acids Res. 25 (1997), 3389-3402; Altschul, J. Mol.Evol. 36 (1993), 290-300; Altschul, J. Mol. Biol. 215 (1990), 403-410),can be used to search for local sequence alignments. BLAST producesalignments of both nucleotide and amino acid sequences to determinesequence similarity. Because of the local nature of the alignments,BLAST is especially useful in determining exact matches or inidentifying similar sequences. The fundamental unit of BLAST algorithmoutput is the High-scoring Segment Pair (HSP). An HSP consists of twosequence fragments of arbitrary but equal lengths whose alignment islocally maximal and for which the alignment score meets or exceeds athreshold or cutoff score set by the user. The BLAST approach is to lookfor HSPs between a query sequence and a database sequence, to evaluatethe statistical significance of any matches found, and to report onlythose matches which satisfy the user-selected threshold of significance.The parameter E establishes the statistically significant threshold forreporting database sequence matches. E is interpreted as the upper boundof the expected frequency of chance occurrence of an HSP (or set ofHSPs) within the context of the entire database search. Any databasesequence whose match satisfies E is reported in the program output.

Analogous computer techniques using BLAST (Altschul (1997), loc. cit.;Altschul (1993), loc. cit.; Altschul (1990), loc. cit.) are used tosearch for identical or related molecules in nucleotide databases suchas GenBank or EMBL. This analysis is much faster than multiplemembrane-based hybridizations. In addition, the sensitivity of thecomputer search can be modified to determine whether any particularmatch is categorized as exact or similar. The basis of the search is theproduct score which is defined as:

$\frac{\% \mspace{11mu} {sequence}\mspace{14mu} {identity} \times \% \mspace{14mu} {maximum}\mspace{14mu} {Blast}\mspace{14mu} {score}}{100}$

and it takes into account both the degree of similarity between twosequences and the length of the sequence match. For example, with aproduct score of 40, the match will be exact within a 1-2% error; and at70, the match will be exact. Similar molecules are usually identified byselecting those which show product scores between 15 and 40, althoughlower scores may identify related molecules.

Moreover, the present invention also relates to nucleic acid moleculesthe sequence of which is degenerate in comparison with the sequence ofan above-described nucleic acid molecules. When used in accordance withthe present invention the term “being degenerate as a result of thegenetic code” means that due to the redundancy of the genetic codedifferent nucleotide sequences code for the same amino acid. Of course,the present invention also envisages the complementary strand to theaforementioned and below mentioned nucleic acid molecules if they may bein a single-stranded form.

Preferably, the nucleic acid molecule according to the invention may beany type of nucleic acid, e.g. DNA, genomicDNA, cDNA, RNA or PNA(peptide nucleic acid).

For the purposes of the present invention, a peptide nucleic acid (PNA)is a polyamide type of DNA analog and the monomeric units for adenine,guanine, thymine and cytosine are available commercially (PerceptiveBiosystems). Certain components of DNA, such as phosphorus, phosphorusoxides, or deoxyribose derivatives, are not present in PNAs. Asdisclosed by Nielsen et al., Science 254:1497 (1991); and Egholm et al.,Nature 365:666 (1993), PNAs bind specifically and tightly tocomplementary DNA strands and are not degraded by nucleases. In fact,PNA binds more strongly to DNA than DNA itself does. This is probablybecause there is no electrostatic repulsion between the two strands, andalso the polyamide backbone is more flexible. Because of this, PNA/DNAduplexes bind under a wider range of stringency conditions than DNA/DNAduplexes, making it easier to perform multiplex hybridization. Smallerprobes can be used than with DNA due to the strong binding. In addition,it is more likely that single base mismatches can be determined withPNA/DNA hybridization because a single mismatch in a PNA/DNA 15-merlowers the melting point (T.sub.m) by 8°-20° C., vs. 4°-16° C. for theDNA/DNA 15-mer duplex. Also, the absence of charge groups in PNA meansthat hybridization can be done at low ionic strengths and reducepossible interference by salt during the analysis.

The DNA may, for example, be genomic DNA or cDNA. The RNA may be, e.g.,mRNA. The nucleic acid molecule may be natural, synthetic orsemisynthetic or it may be a derivative, such as peptide nucleic acid(Nielsen, Science 254 (1991), 1497-1500) or phosphorothioates.Furthermore, the nucleic acid molecule may be a recombinantly producedchimeric nucleic acid molecule comprising any of the aforementionednucleic acid molecules either alone or in combination.

Preferably, the nucleic acid molecule of the present invention is partof a vector. Therefore, the present invention relates in anotherembodiment to a vector comprising the nucleic acid molecule of thisinvention. Such a vector may be, e.g., a plasmid, cosmid, virus,bacteriophage or another vector used e.g. conventionally in geneticengineering, and may comprise further genes such as marker genes whichallow for the selection and/or replication of said vector in a suitablehost cell and under suitable conditions. In a preferred embodiment, saidvector is an expression vector, in which the nucleic acid molecule ofthe present invention is operatively linked and to expression controlsequence(s) allowing expression in prokaryotic or eukaryotic host cellsas described herein. The term “operatively linked”, as used in thiscontext, refers to a linkage between one or more expression controlsequences and the coding region in the polynucleotide to be expressed insuch a way that expression is achieved under conditions compatible withthe expression control sequence.

The nucleic acid molecules of the present invention may thus be insertedinto several commercially available vectors. Nonlimiting examplesinclude plasmid vectors compatible with mammalian cells, such as pUC,pBluescript (Stratagene), pET (Novagen), pREP (Invitrogen), pCRTopo(Invitrogen), pcDNA3 (Invitrogen), pCEP4 (Invitrogen), pMC1 neo(Stratagene), pXT1 (Stratagene), pSG5 (Stratagene), EBO-pSV2neo, pBPV-1,pdBPVMMTneo, pRSVgpt, pRSVneo, pSV2-dhfr, pUCTag, pIZD35, pLXIN and pSIR(Clontech) and pIRES-EGFP (Clontech). Preferably, the nucleic acidmolecules of the present invention are inserted into the vector SignalpIG plus (Ingenius, R&D Systems). Baculovirus vectors such as pBlueBac,BacPacz Baculovirus Expression System (CLONTECH), and MaxBac™Baculovirus Expression System, insect cells and protocols (Invitrogen)are available commercially and may also be used to produce high yieldsof biologically active protein. (see also, Miller (1993), Curr. Op.Genet. Dev., 3, 9; O'Reilly, Baculovirus Expression Vectors: ALaboratory Manual, p. 127). In addition, prokaryotic vectors such aspcDNA2; and yeast vectors such as pYes2 are nonlimiting examples ofother vectors suitable for use with the present invention.

Other preferred expression vectors of the present application are thosefor expressing proteins in Drosophila cells which are well known in theart, such as the DES®-series of Invitrogen. Preferably, said Drosophilacell expression vector is pMTBiP/V5-His B (Invitrogen). ThepMT/BiP/V5-His vector offers the following additional features. It has asmall size (3.6 kb) to improve DNA yields and increase subcloningefficiency, it has a C-terminal V5 epitope tag for rapid detection withAnti-V5 Antibody and it has a C-terminal 6xHis tag for simplepurification of recombinant fusion proteins using nickel-chelatingresin.

For vector modification techniques, see Sambrook and Russel (2001), loc.cit. Vectors can contain one or more replication and inheritance systemsfor cloning or expression, one or more markers for selection in thehost, e.g., antibiotic resistance, and one or more expression cassettes.

The coding sequences inserted in the vector can be synthesized bystandard methods, isolated from natural sources, or prepared as hybrids.Ligation of the coding sequences to transcriptional regulatory elements(e.g., promoters, enhancers, and/or insulators) and/or to other aminoacid encoding sequences can be carried out using established methods.

Furthermore, the vectors may, in addition to the nucleic acid sequencesof the invention, comprise expression control elements, allowing properexpression of the coding regions in suitable hosts. Such controlelements are known to the artisan and may include a promoter,translation initiation codon, translation and insertion site or internalribosomal entry sites (IRES) (Owens, Proc. Natl. Acad. Sci. USA 98(2001), 1471-1476) for introducing an insert into the vector.Preferably, the nucleic acid molecule of the invention is operativelylinked to said expression control sequences allowing expression ineukaryotic or prokaryotic cells.

Control elements ensuring expression in eukaryotic and prokaryotic cellsare well known to those skilled in the art. As mentioned above, theyusually comprise regulatory sequences ensuring initiation oftranscription and optionally poly-A signals ensuring termination oftranscription and stabilization of the transcript. Additional regulatoryelements may include transcriptional as well as translational enhancers,and/or naturally-associated or heterologous promoter regions. Possibleregulatory elements permitting expression in for example mammalian hostcells comprise the CMV-HSV thymidine kinase promoter, SV40, RSV-promoter(Rous sarcome virus), human elongation factor 1α-promoter, CMV enhancer,CaM-kinase promoter or SV40-enhancer.

For the expression in prokaryotic cells, a multitude of promotersincluding, for example, the tac-lac-promoter, the lacUV5 or the trppromoter, has been described. Beside elements which are responsible forthe initiation of transcription such regulatory elements may alsocomprise transcription termination signals, such as SV40-poly-A site orthe tk-poly-A site, downstream of the polynucleotide. In this context,suitable expression vectors are known in the art such as Okayama-BergcDNA expression vector pcDV1 (Pharmacia), pRc/CMV, pcDNA1, pcDNA3(In-Vitrogene, as used, inter alia in the appended examples), pSPORTI(GIBCO BRL) or pGEMHE (Promega), or prokaryotic expression vectors, suchas lambda gt11.

An expression vector according to this invention is at least capable ofdirecting the replication, and preferably the expression, of the nucleicacids and protein of this invention. Suitable origins of replicationinclude, for example, the Col E1, the SV40 viral and the M 13 origins ofreplication. Suitable promoters include, for example, thecytomegalovirus (CMV) promoter, the lacZ promoter, the gal10 promoterand the Autographa californica multiple nuclear polyhedrosis virus(AcMNPV) polyhedral promoter. Suitable termination sequences include,for example, the bovine growth hormone, SV40, lacZ and AcMNPV polyhedralpolyadenylation signals. Examples of selectable markers includeneomycin, ampicillin, and hygromycin resistance and the like.Specifically-designed vectors allow the shuttling of DNA betweendifferent host cells, such as bacteria-yeast, or bacteria-animal cells,or bacteria-fungal cells, or bacteria invertebrate cells.

Beside the nucleic acid molecules of the present invention, the vectormay further comprise nucleic acid sequences encoding for secretionsignals. The secretion signal of the present invention that ispreferably used in accordance with the present invention when thepolypeptide of the present invention is expressed in Drosophila cells,preferably Drosophila S2 cells is the Drosophila BiP secretion signalwell known in the art. The preferred BiP secretion signal that is usedin the context of the present invention is shown in the amino acidsequence of SEQ ID NO: 2 at positions 1 to 28. Other secretion signalsequences are well known to the person skilled in the art. Furthermore,depending on the expression system used leader sequences capable ofdirecting the expressed polypeptide to a cellular compartment may beadded to the coding sequence of the nucleic acid molecules of theinvention and are well known in the art. The leader sequence(s) is (are)assembled in appropriate phase with translation, initiation andtermination sequences, and preferably, a leader sequence capable ofdirecting secretion of translated protein, or a part thereof, into,inter alia, the extracellular membrane. Optionally, the heterologoussequence can encode a fusion protein including an C- or N-terminalidentification peptide imparting desired characteristics, e.g.,stabilization or simplified purification of expressed recombinantproduct. Once the vector has been incorporated into the appropriatehost, the host is maintained under conditions suitable for high levelexpression of the nucleotide sequences, and, as desired, the collectionand purification of the proteins, antigenic fragments or fusion proteinsof the invention may follow. Of course, the vector can also compriseregulatory regions from pathogenic organisms.

Furthermore, said vector may also be, besides an expression vector, agene transfer and/or gene targeting vector. Gene therapy, which is basedon introducing therapeutic genes (for example for vaccination) intocells by ex-vivo or in-vivo techniques is one of the most importantapplications of gene transfer. Suitable vectors, vector systems andmethods for in-vitro or in-vivo gene therapy are described in theliterature and are known to the person skilled in the art; see, e.g.,Giordano, Nature Medicine 2 (1996), 534-539; Schaper, Circ. Res. 79(1996), 911-919; Anderson, Science 256 (1992), 808-813, Isner, Lancet348 (1996), 370-374; Muhlhauser, Circ. Res. 77 (1995), 1077-1086; Wang,Nature Medicine 2 (1996), 714-716; WO 94/29469; WO 97/00957; Schaper,Current Opinion in Biotechnology 7 (1996), 635-640 or Verma, Nature 389(1997), 239-242 and references cited therein.

The nucleic acid molecules of the invention and vectors as describedherein above may be designed for direct introduction or for introductionvia liposomes, or viral vectors (e.g. adenoviral, retroviral) into thecell. Additionally, baculoviral systems or systems based on vacciniavirus or Semliki Forest Virus can be used as eukaryotic expressionsystem for the nucleic acid molecules of the invention. In addition torecombinant production, fragments of the protein, the fusion protein orantigenic fragments of the invention may be produced by direct peptidesynthesis using solid-phase techniques (cf Stewart et al. (1969) SolidPhase Peptide Synthesis; Freeman Co, San Francisco; Merrifield, J. Am.Chem. Soc. 85 (1963), 2149-2154). In vitro protein synthesis may beperformed using manual techniques or by automation. Automated synthesismay be achieved, for example, using Applied Biosystems 431A PeptideSynthesizer (Perkin Elmer, Foster City Calif.) in accordance with theinstructions provided by the manufacturer. Various fragments may bechemically synthesized separately and combined using chemical methods toproduce the full length molecule.

The present invention in addition relates to a host cell geneticallyengineered with the nucleic acid molecule of the invention or a vectorof the present invention. Said host may be produced by introducing saidvector or nucleotide sequence into a host cell which upon its presencein the cell mediates the expression of a protein encoded by thenucleotide sequence of the invention or comprising a nucleotide sequenceor a vector according to the invention wherein the nucleotide sequenceand/or the encoded polypeptide is foreign to the host cell.

By “foreign” it is meant that the nucleotide sequence and/or the encodedpolypeptide is either heterologous with respect to the host, this meansderived from a cell or organism with a different genomic background, oris homologous with respect to the host but located in a differentgenomic environment than the naturally occurring counterpart of saidnucleotide sequence. This means that, if the nucleotide sequence ishomologous with respect to the host, it is not located in its naturallocation in the genome of said host, in particular it is surrounded bydifferent genes. In this case the nucleotide sequence may be eitherunder the control of its own promoter or under the control of aheterologous promoter. The location of the introduced nucleic acidmolecule or the vector can be determined by the skilled person by usingmethods well-known to the person skilled in the art, e.g., SouthernBlotting. The vector or nucleotide sequence according to the inventionwhich is present in the host may either be integrated into the genome ofthe host or it may be maintained in some form extrachromosomally. Inthis respect, it is also to be understood that the nucleotide sequenceof the invention can be used to restore or create a mutant gene viahomologous recombination.

Said host may be any prokaryotic or eukaryotic cell. Suitableprokaryotic/bacterial cells are those generally used for cloning like E.coli, Salmonella typhimurium, Serratia marcescens or Bacillus subtilis.Said eukaryotic host may be a mammalian cell, an amphibian cell, a fishcell, an insect cell, a fungal cell, a plant cell or a bacterial cell(e.g., E coli strains HB101, DH5a, XL1 Blue, Y1090 and JM101).Eukaryotic recombinant host cells are preferred. Examples of eukaryotichost cells include, but are not limited to, yeast, e.g., Saccharomycescerevisiae, Schizosaccharomyces pombe, Kluyveromyces lactis or Pichiapastoris cells, cell lines of human, bovine, porcine, monkey, and rodentorigin, as well as insect cells, including but not limited to,Spodoptera frugiperda insect cells and zebra fish cells.

Drosophila cells, however, are preferred. More preferably, saidDrosophila cells are Drosophila S2 (ATCC CRL-1963) which are, preferablyused for heterologous protein expression in Drosophila expressionsystems, for example, the Drosophila Expression System (DES®). The S2cell line was derived from a primary culture of late stage (20-24 hoursold) Drosophila melanogaster embryos. This versatile cell line growsrapidly at room temperature without CO₂ and is easily adapted tosuspension culture. Generally, when expressing the polypeptide of thepresent invention insect cells are preferred since they have theadvantage that they contain less or, preferably no methylated DNA.Accordingly, when expressing and isolating and preferably purifying thepolypeptide of the present invention, said polypeptide is preferably notcontaminated with methylated DNA to which it can preferably bind.Another advantage of using insect cells is that they grow preferably ina protein-free medium which, thus, minimizes a further contamination ofthe polypeptide of the present invention when isolating, recoveringand/or purifying the polypeptide of the present invention frompreferably culture medium if said polypeptide is preferably secretedinto said culture medium.

Mammalian species-derived cell lines suitable for use and commerciallyavailable include, but are not limited to, L cells, CV-1 cells, COS-1cells (ATCC CRL 1650), COS-7 cells (ATCC CRL 1651), HeLa cells (ATCC CCL2), C1271 (ATCC CRL 1616), BS-C-1 (ATCC CCL 26) and MRC-5 (ATCC CCL171).

In another embodiment, the present invention relates to a method forproducing a polypeptide which is capable of binding methylated DNA,preferably CpG methylated DNA comprising culturing the host cell of theinvention and recovering the produced polypeptide. Said polypeptide ispreferably encoded by a nucleic acid molecule of the invention. Apreferred method for producing the polypeptide on the present inventionis described in Example 2.

The present invention also provides a process for producing cellscapable of expressing a polypeptide of the present invention which iscapable of binding methylated DNA, preferably CpG methylated DNAcomprising genetically engineering cells in vitro by methods known inthe art or by those described herein. Said polypeptide is preferablyencoded by a nucleic acid molecule of the present invention.

A large number of suitable methods exist in the art to producepolypeptides in appropriate hosts. If the host is a unicellular organismor a mammalian or insect cell, the person skilled in the art can revertto a variety of culture conditions that can be further optimized withoutan undue burden of work. Conveniently, the produced protein is harvestedfrom the culture medium or from isolated (biological) membranes byestablished techniques. Furthermore, the produced polypeptide may bedirectly isolated from the host cell.

The polypeptide of the invention may be produced by microbiologicalmethods or by transgenic mammals. It is also envisaged that thepolypeptide of the invention is recovered from transgenic plants.Alternatively, the polypeptide of the invention may be producedsynthetically or semi-synthetically.

For example, chemical synthesis, such as the solid phase proceduredescribed by Houghton Proc. Natl. Acad. Sci. USA (82) (1985), 5131-5135,can be used. Another method is in vitro translation of mRNA. A preferredmethod involves the recombinant production of protein in host cells asdescribed above. For example, nucleotide acid sequences comprising allor a portion of any one of the nucleotide sequences according to theinvention can be synthesized by PCR, inserted into an expression vector,and a host cell transformed with the expression vector. Thereafter, thehost cell is cultured to produce the desired polypeptide, which isisolated and purified. Protein isolation and purification can beachieved by any one of several known techniques; for example and withoutlimitation, ion exchange chromatography, gel filtration chromatographyand affinity chromatography, high pressure liquid chromatography (HPLC),reversed phase HPLC, preparative disc gel electrophoresis. In addition,cell-free translation systems can be used to produce the polypeptides ofthe present invention. Suitable cell-free expression systems for use inaccordance with the present invention include rabbit reticulocytelysate, wheat germ extract, canine pancreatic microsomal membranes, E.coli S30 extract, and coupled transcription/translation systems such asthe TNT-system (Promega). These systems allow the expression ofrecombinant polypeptides or peptides upon the addition of cloningvectors, DNA fragments, or RNA sequences containing coding regions andappropriate promoter elements. As mentioned supra, proteinisolation/purification techniques may require modification of theproteins of the present invention using conventional methods. Forexample, a histidine tag can be added to the protein to allowpurification on a nickel column. Other modifications may cause higher orlower activity, permit higher levels of protein production, or simplifypurification of the protein. After production of the polypeptide of thepresent invention it may be modified by pegylation, derivatization andthe like.

In another embodiment the present invention relates to an antibodyspecifically binding to the polypeptide of the present invention.Preferably, the polypeptide has the cability to bind to methyled DNA andis a bifunctional protein as described herein.

The term “specifically” in this context means that the antibody reactswith the polypeptide of the present invention, but not with onlyportions of said polypeptide, e.g., with the methyl-DNA-binding domain,the Fc portion or a leader or secretion sequence. However, said antibodycould specifically bind to the polypeptide linker of the polypeptide ofthe present invention if such a polypeptide linker is present.

Accordingly, said antibody binds specifically, for example, to a portionof the methyl-DNA-binding domain and the Fc portion of the polypeptideof the present invention or to a portion of the methyl-DNA-bindingdomain and the linker polypeptide or to a portion of the linkerpolypeptide and the Fc portionor as mentioned above, only to the linkerpolypeptide. Whether the antibody specifically reacts as defined hereinabove can easily be tested, inter alia, by comparing the bindingreaction of said antibody with the portions as mentioned above and withonly the respective portion(s) of the polypeptide of the presentinvention.

The antibody of the present invention can be, for example, polyclonal ormonoclonal. The term “antibody” also comprises derivatives or fragmentsthereof which still retain the binding specificity. Techniques for theproduction of antibodies are well known in the art and described, e.g.in Harlow and Lane “Antibodies, A Laboratory Manual”, CSH Press, ColdSpring Harbor, 1988. These antibodies can be used, for example, for theimmunoprecipitation and immunolocalization of the polypeptides of theinvention as well as for the monitoring of the presence of suchpolypeptides, for example, in recombinant organisms or in diagnosis.They can also be used for the identification of compounds interactingwith the proteins according to the invention (as mentioned hereinbelow). For example, surface plasmon resonance as employed in theBIAcore system can be used to increase the efficiency of phageantibodies which bind to an epitope of the polypeptide of the invention(Schier, Human Antibodies Hybridomas 7 (1996), 97-105; Malmborg, J.Immunol. Methods 183 (1995), 7-13).

The present invention furthermore includes chimeric, single chain andhumanized antibodies, as well as antibody fragments, like, inter alia,Fab fragments. Antibody fragments or derivatives further compriseF(ab′)2, Fv or scFv fragments; see, for example, Harlow and Lane, loc.cit. Various procedures are known in the art and may be used for theproduction of such antibodies and/or fragments. Thus, the (antibody)derivatives can be produced by peptidomimetics. Further, techniquesdescribed for the production of single chain antibodies (see, interalia, U.S. Pat. No. 4,946,778) can be adapted to produce single chainantibodies to polypeptide(s) of this invention. Also, transgenic animalsmay be used to express humanized antibodies to polypeptides of thisinvention. Most preferably, the antibody of this invention is amonoclonal antibody. For the preparation of monoclonal antibodies, anytechnique which provides antibodies produced by continuous cell linecultures can be used. Examples for such techniques include the hybridomatechnique (Köhler and Milstein Nature 256 (1975), 495-497), the triomatechnique, the human B-cell hybridoma technique (Kozbor, ImmunologyToday 4 (1983), 72) and the EBV-hybridoma technique to produce humanmonoclonal antibodies (Cole et al., Monoclonal Antibodies and CancerTherapy, Alan R. Liss, Inc. (1985), 77-96). Techniques describing theproduction of single chain antibodies (e.g., U.S. Pat. No. 4,946,778)can be adapted to produce single chain antibodies to immunogenicpolypeptides as described above. Furthermore, transgenic mice may beused to express humanized antibodies directed against said immunogenicpolypeptides. It is in particular preferred that the antibodies/antibodyconstructs as well as antibody fragments or derivatives to be employedin accordance with this invention or capable to be expressed in a cell.This may, inter alia, be achieved by direct injection of thecorresponding proteineous molecules or by injection of nucleic acidmolecules encoding the same. Furthermore, gene therapy approaches areenvisaged.

Accordingly, in context of the present invention, the term “antibodymolecule” relates to full immunoglobulin molecules as well as to partsof such immunoglobulin molecules. Furthermore, the term relates, asdiscussed above, to modified and/or altered antibody molecules, likechimeric and humanized antibodies. The term also relates to monoclonalor polyclonal antibodies as well as to recombinantly or syntheticallygenerated/synthesized antibodies. The term also relates to intactantibodies as well as to antibody fragments thereof, like, separatedlight and heavy chains, Fab, Fab/c, Fv, Fab′, F(ab′)2. The term“antibody molecule” also comprises bifunctional antibodies and antibodyconstructs, like single chain Fvs (scFv) or antibody-fusion proteins. Itis also envisaged in context of this invention that the term “antibody”comprises antibody constructs which may be expressed in cells, e.g.antibody constructs which may be transfected and/or transduced via,inter alia, viruses or vectors. Of course, the antibody of the presentinvention can be coupled, linked or conjugated to detectable substancesas described herein above in connection with the Fc portion of thepolypeptide of the present invention.

The present invention also provides a composition comprising the nucleicacid molecule, the vector, the host cell, the polypeptide or theantibody of the present invention.

The term “composition”, as used in accordance with the presentinvention, relates to (a) composition(s) which comprise(s) at least oneof the aforementioned compounds. It is envisaged that the compositionsof the present invention which are described herein below comprise theaforementioned compounds in any combination. It may, optionally,comprise further molecules which are capable of binding methylated DNA,preferably CpG methylated DNA. The composition may be in solid, liquidor gaseous form and may be, inter alia, in the form of (a) powder(s),(a) tablet(s), (a) solution(s) (an) aerosol(s), granules, pills,suspensions, emulsions, capules, syrups, liquids, elixirs, extracts,tincture or fluid extracts or in a form which is particularly suitablefor oral or parental or topic administration.

Additionally, the present invention relates to a kit comprising thenucleic acid molecule, the vector, the host, the polypeptide or theantibody of the present invention.

Advantageously, the kit of the present invention further comprises,optionally (a) reaction buffer(s), storage solutions and/or remainingreagents or materials required for the conduct of scientific ordiagnostic assays or the like. Furthermore, parts of the kit of theinvention can be packaged individually in vials or bottles or incombination in containers or multicontainer units.

The kit of the present invention may be advantageously used, inter alia,for carrying out the method for isolating, enriching, purifying and/ordetecting methylated DNA as described herein and/or it could be employedin a variety of applications referred herein, e.g., as diagnostic kits,as research tools or therapeutic tools. Additionally, the kit of theinvention may contain means for detection suitable for scientific,medical and/or diagnostic purposes. The manufacture of the kits followspreferably standard procedures which are known to the person skilled inthe art.

As described above, the present invention is based on the surprisingfinding that a bifunctional, antibody-like molecule comprising amethyl-DNA-binding domain and an Fc portion of an antibody is able tospecifically bind methylated DNA, preferably CpG methylated DNA withhigh affinity and high avidity which renders it a suitable diagnostictool for isolating, enriching and/or detecting methylated DNA from morethan 10 ng, less than 10 ng, less than 7.5 ng, less than 5 ng, less than2.5 ng or from about 1 ng in a sample.

Accordingly, in a preferred embodiment the composition according to theinvention is a diagnostic composition, optionally further comprisingsuitable means for detection.

A further embodiment of the present invention is the use of thepolypeptide of the present invention for the detection of methylatedDNA.

In addition, the nucleic acid molecules, the polypeptide, the vector,the host cell or the antibody of the present invention are used for thepreparation of a diagnostic composition for detecting methylated DNA.

Moreover, the nucleic acid molecules, the polypeptide, the vector, thehost cell or the antibody of the present invention are used for thepreparation of a diagnostic composition for the detection of tumoroustissue or tumor cells.

As mentioned herein, the polypeptide of the present invention hasunexpected superior properties, in particular for isolating, enriching,purifying and/or detecting methylated DNA, preferably CpG methylatedDNA. Thus, the present invention provides various diagnostic uses andmethods employing the polypeptide of the present invention. A preferredsmall scale enrichment procedure of methylated DNA, preferably CpGmethylated DNA is described in Example 3. Briefly, the polypeptide ofthe present invention is, for example, bound to Protein A sepharose andwashed to remove inbound protein. Next, DNA of interest is preferablydigested and added to the bound polypeptide of the present invention.Furthermore, said digested DNA is incubated with the bound polypeptideof the present invention, washed and, after having been bound by thepolypeptide of the present invention is eluted.

Accordingly, the present invention relates also to an in vitro methodfor detecting methylated DNA comprising

-   (a) contacting a sample comprising methylated and/or unmethylated    DNA with the polypeptide of the present invention; and-   (b) detecting the binding of said polypeptide to methylated DNA.

Preferably, said in vitro method is reverse South-Western blotting asexemplified in Example 3, immune precipitation, affinity purification ofmethylated DNA or Methyl-CpG-immunoprecipitation (MCIp) as exemplifiedin Example 4 and 5. However, said in vitro method is not limitedthereto, but could basically be any procedure in which the polypeptideof the present invention is linked to a solid matrix, for example, amatrix such as sepharose, agarose, capillaries, vessel walls, as is alsodescribed herein in connection with the diagnostic composition of thepresent invention.

More preferably, the aforementioned in vitro methods further comprise asstep (c) analyzing the methylated DNA, for example, by sequencing,Southern Blot, restriction enzyme digestion, bisulfite sequencing,pyrosequencing or PCR. Yet, analyzing methylated DNA which has beenisolated, enriched, purified and/or detected by using the polypeptide ofthe present invention is not limited to the aforementioned methods, butencompasses all methods known in the art for analyzing methylated DNA,e.g. RDA, microarrays and the like.

A preferred diagnostic application of the polypeptide of the presentinvention is the so-called MB-PCR shown in FIG. 7. Briefly, in a firststep the polypeptide of the present invention is added into a coatablePCR-vessel, for example, TopYield Strips from Nunc. In doing so, thepolypeptide is preferably coated onto the inner surface of said vesselby techniques known in the art. In a next step, blocking reagents, e.g.,4.5% milk powder is added into the coated PCR vessel. In a further step,preferably DNA-fragments of interest (for example, methylated and/orunmethylated DNA-fragments) are added into the coated and blocked PCRvessel. It is believed that the polypeptide of the present inventionbinds specifically to methylated DNA, if present. In a following step,the coated and blocked PCR vessel containing preferably DNA-fragments isincubated and then washed to remove unbound DNA-fragments. Afterwards, aPCR mix including preferably gene-specific primers or, but alsopreferred, at least two, three, four, five, six, seven etc. pairs ofprimers for, e.g., multiplex PCR for the gene or genlocus or genloci ofinterest which is/are suspected to be methylated or unmethylated isadded to run preferably, a real time PCR or conventional PCR followed bygelelectrophoresis to separate amplification products.

MB-PCR is preferably done as follows:

Preferably, the PCR tubes are prepared using heat stable TopYield™Strips (Nunc Cat. No. 248909). Preferably, 50 μl of the polypeptide ofthe present invention, preferably, in a recombinant form (diluted at 15μg/ml in 10 mM Tris/HCl pH 7.5) were added to each well and incubatedovernight at 4° C. Preferably, wells are washed three times with 200 μlTBS (20 mM Tris, pH 7.4 containing 150 mM NaCl) and blocked overnight at4 C with 100 μl Blocking Solution (10 mM Tris, pH 7.5 containing 150 mMNaCl, 4.5% skim milk powder, 5 mM EDTA and 0.8 μg/ml of each polyd(I/C), poly d(A/T) and poly d(CG)). Preferably, tubes are then washedthree times with 200 μl TBST (TBS containing 0.1% Tween-20).

Preferably, 50 μl Binding Buffer (20 mM Tris, pH 7.5 containing 400 mMNaCl, 2 mM MgCl₂, 0.5 mM EDTA, and 0.1% Tween-20) are added to each welland preferably 1 μl of digested DNA, preferably genomic DNA digestedwith MseI in an amount of preferably 10 ng/μl is added to every secondwell (M-reaction).

Genomic DNA is preferably prepared by using a kit known in the art, forexample, using Blood and Cell Culture Midi Kit (Qiagen). The quality ofthe genomic DNA-preparation is preferably controlled by agarose gelelectrophoresis and DNA concentration was preferably determined by UVspectrophotometry. Quantitation of DNA is preferably done by usingPicoGreen dsDNA Quantitation Reagent (Molecular Probes).

The wells containing the polypeptide of the present invention and DNA,preferably DNA-fragments (generated by enzymatic digestion ormechanically fragmented) are incubated on a shaker at preferably 4° C.for preferably 3 hours. Preferably, tubes were washed three times with200 μl Binding Buffer and once with 10 mM Tris/HCl pH 7.5.

Next, PCR was preferably carried out directly in the TopYield™ Strips.Preferably, the PCR-Mix (50 μl/well) contained a standard PCR buffer(Roche), preferably 2.5 U FastStart Taq DNA Polymerase (Roche),preferably 10 pmol of each gene-specific primer (synthesized by Qiagen),dNTPs (preferably 200 mM each, Amersham/Pharmacia) preferably 1 Mbetaine (Sigma), primer sequences and cycling parameters for specificgenes of interest are shown in Tables 2 and 3 in Example 6. Of course,any other suitable gene specific or genlocus specific or genlocispecific primers can be designed by the person skilled in the art.Moreover, the skilled artisan can readily determine and/or test the PCRparameters most suitable for the primer(s) and gene(s), genlocus/genlociof interest. After adding the PCR-mix, preferably 1 μl Mse I-digestedDNA (preferably in an amount of 10 ng/μl) is added to every second otherwell, that was not previously incubated with DNA-fragments (P-reaction).Preferably, PCR-products are analysed using agarose gel electrophoresisand the ethidium bromide stained gel was scanned using, for example, aTyphoon 9200 Imager (Amersham/Pharmacia).

Accordingly, it is envisaged that the polypeptide of the presentinvention is useful for the detection of methylated DNA, preferablyCpG-methylated DNA in a sample as described herein below which mayinclude (a) single cell(s). It is also envisaged to be useful for wholecells. “Whole cell” means the genomic context of a whole single cell.Thus, it could be useful for a genome-wide analysis of methylated DNA.

Such a method comprises preferably an enriching/purifying step ofmethylated DNA using the polypeptide of the present invention and adetection step, e.g., hybridization of genomic DNA microarrays, tilingarrays, low-density arrays or lab-on-a-chip-approaches. The personskilled in the art is readily in a position to carry out the detectionmethods which are known in the art. Some of them are shown in theappended Examples, wherein the polypeptide of the present invention isused for enriching, purifying and/or isolating methylated DNA. OneExample shows a so-called MB-PCR which may be suitable forhigh-throughput, robust one-tube assays. Furthermore, the polypeptide ofthe present invention may be particularly useful in the detection ofCpG-methylation on single gene level. Such a method preferably comprisesthe step of enriching and/or purifying methylated DNA, preferablyCpG-methylated DNA of a single gene and the step of detecting saidmethylated DNA by employing PCR, real-time PCR and the like.

Another possible diagnostic application of the polypeptide of thepresent invention is immunohistochemistry. Accordingly, the polypeptideof the present invention can be used to “stain” methylated DNA,preferably CpG-methylated DNA. Either the polypeptide of the presentinvention is via its Fc portion coupled, linked or conjugated to asuitable detectable substance as described herein or, for example, asecond anti-Fc portion antibody is used for detecting the polypeptide ofthe present invention when bound to methylated DNA.

It is assumed that some malignancies can be detected by the methods ofthe present invention by their methylation pattern/profile which may,thus, be of a prognostic and/or predicable value. That means that themethylation pattern can be used for setting up a pharmacologic profilefor a patient. For example, the susceptibility and/or sensitivity to,e.g., anti-cancer drug may be determined if it is detected that certainoncogenes and/or tumor suppressor genes are either hyper- orhypomethylated. Accordingly, the skilled artisan chooses the mostappropriate medicament to avoid negative and/or adverse effects if, forexample, said medicament may inhibit oncogenes although said oncogenesare already hypermethylated and, thus, assumed to be inactive.

The herein described methods may be useful for, firstly, identifyinggenloic and/or genes which are hyper- or mypomethylated in a malignancysuch as cancer or a tumorous disease and, secondly, provide the basisfor assaying the methylation status of such genloci and/or genes on asingle gene level. Said malignancies are preferably tumors The tumor canany possible type of tumor. Examples are skin, breast, brain, cervicalcarcinomas, testicular carcinomas, head and neck, lung, mediastinum,gastrointestinal tract, genitourinary system, gynaecological system,breast, endocrine system, skin, childhood, unknown primary site ormetastatic cancer, a sarcoma of the soft tissue and bone, amesothelioma, a melanoma, a neoplasm of the central nervous system, alymphoma, a leukaemia, a paraneoplastic syndrome, a peritonealcarcinomastosis, a immunosuppression-related malignancy and/ormetastatic cancer etc. The tumor cells may, e.g., be derived from: headand neck, comprising tumors of the nasal cavity, paranasal sinuses,nasopharynx, oral cavity, oropharynx, larynx, hypopharynx, salivaryglands and paragangliomas, a cancer of the lung, comprising non-smallcell lung cancer, small cell lung cancer, a cancer of the mediastinum, acancer of the gastrointestinal tract, comprising cancer of theoesophagus, stomach, pancreas, liver, biliary tree, small intestine,colon, rectum and anal region, a cancer of the genitourinary system,comprising cancer of the kidney, urethra, bladder, prostate, urethra,penis and testis, a gynaecologic cancer, comprising cancer of thecervix, vagina, vulva, uterine body, gestational trophoblastic diseases,ovarian, fallopian tube, peritoneal, a cancer of the breast, a cancer ofthe endocrine system, comprising a tumor of the thyroid, parathyroid,adrenal cortex, pancreatic endocrine tumors, carcinoid tumor andcarcinoid syndrome, multiple endocrine neoplasias, a sarcoma of the softtissue and bone, a mesothelioma, a cancer of the skin, a melanoma,comprising cutaneous melanomas and intraocular melanomas, a neoplasm ofthe central nervous system, a cancer of the childhood, comprisingretinoblastoma, Wilm's tumor, neurofibromatoses, neuroblastoma, Ewing'ssarcoma family of tumors, rhabdomyosarcoma, a lymphoma, comprisingnon-Hodgkin's lymphomas, cutaneous T-cell lymphomas, primary centralnervous system lymphoma, and Hodgkin's disease, a leukaemia, comprisingacute leukemias, chronic myelogenous and lymphocytic leukemias, plasmacell neoplasms and myelodysplastic syndromes, a paraneoplastic syndrome,a cancer of unknown primary site, a peritoneal carcinomastosis, aimmunosuppression-related malignancy, comprising AIDS-relatedmalignancies, comprising Kaposi's sarcoma, AIDS-associated lymphomas,AIDS-associated primary central nervous system lymphoma, AIDS-associatedHodgkin's disease and AIDS-associated anogenital cancers, andtransplantation-related malignancies, a metastatic cancer to the liver,metastatic cancer to the bone, malignant pleural and pericardialeffusions and malignant ascites. It is mostly preferred that said canceror tumorous disease is cancer of the head and neck, lung, mediastinum,gastrointestinal tract, genitourinary system, gynaecological system,breast, endocrine system, skin, childhood, unknown primary site ormetastatic cancer, a sarcoma of the soft tissue and bone, amesothelioma, a melanoma, a neoplasm of the central nervous system, alymphoma, a leukemia, a paraneoplastic syndrome, a peritonealcarcinomastosis, a immunosuppression-related malignancy and/ormetastatic cancer. Preferred tumors are AML, plasmacytoma or CLL.

The diagnostic composition of the present invention comprises at leastone of the herein described compounds of the invention. The diagnosticcomposition may be used, inter alia, for methods for isolating,enriching and/or determining the presence of methylated DNA, preferablyCpG methylated DNA, for example, in a sample from an individual asdescribed above.

In accordance with the present invention by the term “sample” isintended any biological sample obtained from an individual, cell line,tissue culture, or other source containing polynucleotides orpolypeptides or portions thereof. As indicated, biological samplesinclude body fluids (such as blood, sera, plasma, urine, synovial fluidand spinal fluid) and tissue sources found to express thepolynucleotides of the present invention. Methods for obtaining tissuebiopsies and body fluids from mammals are well known in the art. Abiological sample which includes genomic DNA, mRNA or proteins ispreferred as a source.

Further applications of the diagnostic compositions are described hereinand are shown in the appended Examples.

The diagnostic composition optionally comprises suitable means fordetection. The nucleic acid molecule(s), vector(s), host(s),antibody(ies), and polypeptide(s) described above are, for example,suitable for use in immunoassays in which they can be utilized in liquidphase or bound to a solid phase carrier. Examples of well-known carriersinclude glass, polystyrene, polyvinyl ion, polypropylene, polyethylene,polycarbonate, dextran, nylon, amyloses, natural and modifiedcelluloses, polyacrylamides, agaroses, and magnetite. The nature of thecarrier can be either soluble or insoluble for the purposes of theinvention.

Solid phase carriers are known to those in the art and may comprisepolystyrene beads, latex beads, magnetic beads, colloid metal particles,glass and/or silicon chips and surfaces, nitrocellulose strips,membranes, sheets, duracytes and the walls of wells of a reaction tray,plastic tubes or other test tubes. Suitable methods of immobilizingnucleic acid molecule(s), vector(s), host(s), antibody(ies), aptamer(s),polypeptide(s), etc. on solid phases include but are not limited toionic, hydrophobic, covalent interactions or (chemical) crosslinking andthe like. Examples of immunoassays which can utilize said compounds ofthe invention are competitive and non-competitive immunoassays in eithera direct or indirect format. Commonly used detection assays can compriseradioisotopic or non-radioisotopic methods. Examples of suchimmunoassays are the radioimmunoassay (RIA), the sandwich (immunometricassay) and the Northern or Southern blot assay. Furthermore, thesedetection methods comprise, inter alia, IRMA (Immune RadioimmunometricAssay), EIA (Enzyme Immuno Assay), ELISA (Enzyme Linked Immuno Assay),FIA (Fluorescent Immuno Assay), and CLIA (Chemioluminescent ImmuneAssay). Furthermore, the diagnostic compounds of the present inventionmay be are employed in techniques like FRET (Fluorescence ResonanceEnergy Transfer) assays.

Appropriate labels and methods for labeling are known to those ofordinary skill in the art. Examples of the types of labels which can beused in the present invention include inter alia, fluorochromes (likefluorescein, rhodamine, Texas Red, etc.), enzymes (like horse radishperoxidase, β-galactosidase, alkaline phosphatase), radioactive isotopes(like ³²P, ³³P, ³⁵S or ¹²⁵I), biotin, digoxygenin, colloidal metals,chemi- or bioluminescent compounds (like dioxetanes, luminol oracridiniums).

A variety of techniques are available for labeling biomolecules, arewell known to the person skilled in the art and are considered to bewithin the scope of the present invention and comprise, inter alia,covalent coupling of enzymes or biotinyl groups, phosphorylations,biotinylations, random priming, nick-translations, tailing (usingterminal transferases). Such techniques are, e.g., described in Tijssen,“Practice and theory of enzyme immunoassays”, Burden and von Knippenburg(Eds), Volume 15 (1985); “Basic methods in molecular biology”, Davis LG, Dibmer M D, Battey Elsevier (1990); Mayer, (Eds) “Immunochemicalmethods in cell and molecular biology” Academic Press, London (1987); orin the series “Methods in Enzymology”, Academic Press, Inc. Detectionmethods comprise, but are not limited to, autoradiography, fluorescencemicroscopy, direct and indirect enzymatic reactions, etc.

Another preferred composition of the present invention is apharmaceutical composition optionally further comprising apharmaceutical acceptable carrier. Said pharmaceutical compositioncomprises, inter alia, the polypeptide of the present invention whichmay be coupled to a further polypeptide, for example, a histonedeacetylase, a histone acetylase, DNA-methylase and/or DNA-demethylase.It could also be coupled with a restriction enzyme or a ribozyme. It isbelieved that if the polypeptide of the present invention coupled withone or more further proteian as described above binds to methylated DNA,it may target said further protein(s) to DNA. Accordingly, aDNA-methylase could hyper-methylate a hypomethylated DNA, for example, ahypomethylated oncogenic locus or oncogene or a DNA. In doing so, geneinactivation could be achieved.

Alternatively, a DNA-demethylase may demethylate a hypermethylated geneor genlocus, for example, a tumor suppressor gene or genlocus. In doingso, gene activation could be achieved.

A histone deacetylase contribute to transcriptional repression of anactive gene by deacetylating acetylated lysine residues of histones,thereby leading to a tighter packaging of DNA to histones and, generepression. A histone acetylase could do the contrary effect as is knownin the art.

A restriction enzyme or a ribozyme could exert its effect when targetedto DNA which should be cleaved. Appropriate restriction enzymes areknown in the art. Ribozymes specific for target-DNA sequences can beprepared as is known in the art.

Accordingly, the pharmaceutical composition could be useful for treatingcancer and/or tumorous disease. Both of which are known to be caused byuncontrolled gene expression, activation and/or repression which is,inter alia, regulated by histone acetylation/deacetylation and/orDNA-methylation/demethylation.

The pharmaceutical composition may be administered with aphysiologically acceptable carrier to a patient, as described herein. Ina specific embodiment, the term “pharmaceutically acceptable” meansapproved by a regulatory agency or other generally recognizedpharmacopoeia for use in animals, and more particularly in humans. Theterm “carrier” refers to a diluent, adjuvant, excipient, or vehicle withwhich the therapeutic is administered. Such pharmaceutical carriers canbe sterile liquids, such as water and oils, including those ofpetroleum, animal, vegetable or synthetic origin, such as peanut oil,soybean oil, mineral oil, sesame oil and the like. Water is a preferredcarrier when the pharmaceutical composition is administeredintravenously. Saline solutions and aqueous dextrose and glycerolsolutions can also be employed as liquid carriers, particularly forinjectable solutions. Suitable pharmaceutical excipients include starch,glucose, lactose, sucrose, gelatin, malt, rice, flour, chalk, silicagel, sodium stearate, glycerol monostearate, talc, sodium ion, driedskim milk, glycerol, propylene, glycol, water, ethanol and the like. Thecomposition, if desired, can also contain minor amounts of wetting oremulsifying agents, or pH buffering agents. These compositions can takethe form of solutions, suspensions, emulsion, tablets, pills, capsules,powders, sustained-release formulations and the like. The compositioncan be formulated as a suppository, with traditional binders andcarriers such as triglycerides. Oral formulation can include standardcarriers such as pharmaceutical grades of mannitol, lactose, starch,magnesium stearate, sodium saccharine, cellulose, magnesium carbonate,etc. Examples of suitable pharmaceutical carriers are described in“Remington's Pharmaceutical Sciences” by E. W. Martin. Such compositionswill contain a therapeutically effective amount of the aforementionedcompounds, preferably in purified form, together with a suitable amountof carrier so as to provide the form for proper administration to thepatient. The formulation should suit the mode of administration.

In another preferred embodiment, the composition is formulated inaccordance with routine procedures as a pharmaceutical compositionadapted for intravenous administration to human beings. Typically,compositions for intravenous administration are solutions in sterileisotonic aqueous buffer. Where necessary, the composition may alsoinclude a solubilizing agent and a local anesthetic such as lidocaine toease pain at the site of the injection. Generally, the ingredients aresupplied either separately or mixed together in unit dosage form, forexample, as a dry lyophilised powder or water free concentrate in ahermetically sealed container such as an ampoule or sachette indicatingthe quantity of active agent. Where the composition is to beadministered by infusion, it can be dispensed with an infusion bottlecontaining sterile pharmaceutical grade water or saline. Where thecomposition is administered by injection, an ampoule of sterile waterfor injection or saline can be provided so that the ingredients may bemixed prior to administration.

The pharmaceutical composition of the invention can be formulated asneutral or salt forms. Pharmaceutically acceptable salts include thoseformed with anions such as those derived from hydrochloric, phosphoric,acetic, oxalic, tartaric acids, etc., and those formed with cations suchas those derived from sodium, potassium, ammonium, calcium, ferrichydroxides, isopropylamine, triethylamine, 2-ethylamino ethanol,histidine, procaine, etc.

In vitro assays may optionally be employed to help identify optimaldosage ranges. The precise dose to be employed in the formulation willalso depend on the route of administration, and the seriousness of thedisease or disorder, and should be decided according to the judgment ofthe practitioner and each patient's circumstances. Effective doses maybe extrapolated from dose-response curves derived from in vitro oranimal model test systems. Preferably, the pharmaceutical composition isadministered directly or in combination with an adjuvant.

The pharmaceutical composition is preferably designed for theapplication in gene therapy. The technique of gene therapy has alreadybeen described above in connection with the nucleic acid molecules ofthe invention and all what has been said there also applies inconnection with the pharmaceutical composition. For example, the nucleicacid molecule in the pharmaceutical composition is preferably in a formwhich allows its introduction, expression and/or stable integration intocells of an individual to be treated.

For gene therapy, various viral vectors which can be utilized, forexample, adenovirus, herpes virus, vaccinia, or, preferably, an RNAvirus such as a retrovirus. Examples of retroviral vectors in which asingle foreign gene can be inserted include, but are not limited to:Moloney murine leukemia virus (MoMuLV), Harvey murine sarcoma virus(HaMuSV), murine mammary tumor virus (MuMTV), and Rous Sarcoma Virus(RSV). A number of additional retroviral vectors can also incorporatemultiple genes. All of these vectors can transfer or incorporate a genefor a selectable marker so that transduced cells can be identified andgenerated. Retroviral vectors can be made target specific by inserting,for example, a polynucleotide encoding a sugar, a glycolipid, or aprotein. Those of skill in the art will know of, or can readilyascertain without undue experimentation, specific polynucleotidesequences which can be inserted into the retroviral genome to allowtarget specific delivery of the retroviral vector containing theinserted polynucleotide sequence.

Since recombinant retroviruses are preferably defective, they requireassistance in order to produce infectious vector particles. Thisassistance can be provided, for example, by using helper cell lines thatcontain plasmids encoding all of the structural genes of the retrovirusunder the control of regulatory sequences within the LTR. These plasmidsare missing a nucleotide sequence which enables the packaging mechanismto recognize an RNA transcript for encapsidation. Helper cell lineswhich have deletions of the packaging signal include, but are notlimited to w2, PA317 and PA12, for example. These cell lines produceempty virions, since no genome is packaged. If a retroviral vector isintroduced into such cells in which the packaging signal is intact, butthe structural genes are replaced by other genes of interest, the vectorcan be packaged and vector virion produced. Alternatively, NIH 3T3 orother tissue culture cells can be directly transfected with plasmidsencoding the retroviral structural genes gag, pol and env, byconventional calcium phosphate transfection. These cells are thentransfected with the vector plasmid containing the genes of interest.The resulting cells release the retroviral vector into the culturemedium. Another targeted delivery system for the nucleic acid moleculesof the present invention is a colloidal dispersion system. Colloidaldispersion systems include macromolecule complexes, nanocapsules,microspheres, beads, and lipid-based systems including oil-in-wateremulsions, micelles, mixed micelles, and liposomes. The preferredcolloidal system of this invention is a liposome. Liposomes areartificial membrane vesicles which are useful as delivery vehicles invitro and in vivo. It has been shown that large unilamellar vesicles(LUV), which range in size from 0.2-4.0 pm can encapsulate a substantialpercentage of an aqueous buffer containing large macromolecules. RNA,DNA and intact virions can be encapsulated within the aqueous interiorand be delivered to cells in a biologically active form (Fraley, et al.,Trends Biochem. Sci., 6:77, 1981). In addition to mammalian cells,liposomes have been used for delivery of polynucleotides in plant, yeastand bacterial cells. In order for a liposome to be an efficient genetransfer vehicle, the following characteristics should be present: (1)encapsulation of the genes of interest at high efficiency while notcompromising their biological activity; (2) preferential and substantialbinding to a target cell in comparison to non-target cells; (3) deliveryof the aqueous contents of the vesicle to the target cell cytoplasm athigh efficiency; and (4) accurate and effective expression of geneticinformation (Mannino, et al., Biotechniques, 6:682, 1988). Thecomposition of the liposome is usually a combination of phospholipids,particularly high-phase-transition-temperature phospholipids, usually incombination with steroids, especially cholesterol. Other phospholipidsor other lipids may also be used. The physical characteristics ofliposomes depend on pH, ionic strength, and the presence of divalentcations. Examples of lipids useful in liposome production includephosphatidyl compounds, such as phosphatidylglycerol,phosphatidylcholine, phosphatidylserine, phosphatidylethanolamine,sphingolipids, cerebrosides, and gangliosides. Particularly useful arediacylphosphatidylglycerols, where the lipid moiety contains from 14-18carbon atoms, particularly from 16-18 carbon atoms, and is saturated.Illustrative phospholipids include egg phosphatidylcholine,dipalmitoylphosphatidylcholine and distearoylphosphatidylcholine. Thetargeting of liposomes can be classified based on anatomical andmechanistic factors. Anatomical classification is based on the level ofselectivity, for example, organ-specific, cell-specific, andorganelle-specific. Mechanistic targeting can be distinguished basedupon whether it is passive or active. Passive targeting utilizes thenatural tendency of liposomes to distribute to cells of thereticulo-endothelial system (RES) in organs which contain sinusoidalcapillaries.

In a preferred embodiment, the compositions of the present invention maybe useful for in vivo imaging methylated DNA, preferably CpG methylatedDNA. Accordingly said composition is administered to a subject in needthereof. In the context of the present invention the term “subject”means an individual in need of a treatment of an affective disorder.Preferably, the subject is a vertebrate, even more preferred a mammal,particularly preferred a human. The term “administered” meansadministration of a therapeutically or diagnostically effective dose ofthe aforementioned nucleic acid molecule encoding the polypeptide of thepresent invention to an individual. By “therapeutically ordiagnostically effective amount” is meant a dose that produces theeffects for which it is administered. The exact dose will depend on thepurpose of the treatment or diagnosis, and will be ascertainable by oneskilled in the art using known techniques. As is known in the art anddescribed above, adjustments for systemic versus localized delivery,age, body weight, general health, sex, diet, time of administration,drug interaction and the severity of the condition may be necessary, andwill be ascertainable with routine experimentation by those skilled inthe art. The methods are applicable to both human therapy and veterinaryapplications. The compounds described herein having the desiredtherapeutic activity may be administered in a physiologically acceptablecarrier to a patient, as described herein. Depending upon the manner ofintroduction, the compounds may be formulated in a variety of ways asdiscussed below. The concentration of therapeutically active compound inthe formulation may vary from about 0.1-100 wt %. The agents maybeadministered alone or in combination with other treatments.

The administration of the pharmaceutical composition can be done in avariety of ways as discussed above, including, but not limited to,orally, subcutaneously, intravenously, intra-arterial, intranodal,intramedullary, intrathecal, intraventricular, intranasally,intrabronchial, transdermally, intranodally, intrarectally,intraperitoneally, intramuscularly, intrapulmonary, vaginally, rectally,or intraocularly. In some instances, for example, in the treatment ofwounds and inflammation, the candidate agents may be directly applied asa solution dry spray.

The attending physician and clinical factors will determine the dosageregimen. As is well known in the medical arts, dosages for any onepatient depends upon many factors, including the patient's size, bodysurface area, age, the particular compound to be administered, sex, timeand route of administration, general health, and other drugs beingadministered concurrently. A typical dose can be, for example, in therange of 0.001 to 1000 μg; however, doses below or above this exemplaryrange are envisioned, especially considering the aforementioned factors.

The dosages are preferably given once a week, however, duringprogression of the treatment the dosages can be given in much longertime intervals and in need can be given in much shorter time intervals,e.g., daily. In a preferred case the immune response is monitored usingherein described methods and further methods known to those skilled inthe art and dosages are optimized, e.g., in time, amount and/orcomposition. Dosages will vary but a preferred dosage for intravenousadministration of DNA is from approximately 10⁶ to 10¹² copies of theDNA molecule. If the regimen is a continuous infusion, it should also bein the range of 1 μg to 10 mg units per kilogram of body weight perminute, respectively. Progress can be monitored by periodic assessment.The pharmaceutical composition of the invention may be administeredlocally or systemically. Administration will preferably be parenterally,e.g., intravenously. Preparations for parenteral administration includesterile aqueous or non-aqueous solutions, suspensions, and emulsions.Examples of non-aqueous solvents are propylene glycol, polyethyleneglycol, vegetable oils such as olive oil, and injectable organic esterssuch as ethyl oleate. Aqueous carriers include water, alcoholic/aqueoussolutions, emulsions or suspensions, including saline and bufferedmedia. Parenteral vehicles include sodium ion solution, Ringer'sdextrose, dextrose and sodium ion, lactated Ringer's, or fixed oils.Intravenous vehicles include fluid and nutrient replenishers,electrolyte replenishers (such as those based on Ringer's dextrose), andthe like. Preservatives and other additives may also be present such as,for example, antimicrobials, anti-oxidants, chelating agents, and inertgases and the like.

It is also envisaged that the pharmaceutical compositions are employedin co-therapy approaches with other agents, for example, useful indetecting methylated DNA and, thus, for example, useful in diagnosingmalignancies which may show a typical methylated pattern.

The figures show:

FIG. 1: FIG. 1 shows the nucleotide sequence of plasmid pMTBip/MBD2-Fc(SEQ ID NO:1) and the protein sequence (in bold; see also SEQ ID NO: 2)of the MBD2-Fc bifunctional protein which is encoded by plasmidpMTBip/MBD2-Fc.

-   -   The amino acid sequence of the MBD2-Fc bifunctional protein has        the following features.    -   AA 1-28 (nt 851-934): Drosophila BiP secretion signal (leader        peptide from pMT/Bip/V5-His vector)    -   AA 29-115 (nt 935-1196): AA 144-230 of human MBD2    -   AA 116-129 (nt 1196-1237): Flexible Linker (AAADPIEGRGGGGG)        (amino acids 116-129 of SEQ ID NO: 2)    -   AA 130-361 (nt 1238-1933): AA 99-330 of human IGHG1

FIG. 2: Expression of MBD2-F_(c) in Drosophila Schneider-cells. Stablytransfected S2 cells were seeded in Medium w/o FCS, with and w/o 500 μMCuSO₄. The supernatant was collected after 4 days and precleared o/n at4° C. using sepharose beads. 1 ml precleared supernatant wasprecipitated using protein A sepharose, washed, resuspended inSDS-loading dye and subjected to SDS-PAGE. The gel was Coomassie-stainedto detect precipitated protein.

FIG. 3: Reverse South-Western Blot. A 650 by PCR-fragment of humanICSBP-promoter (A) or methylated promoter fragments (50 ng) of varyingCpG-density (B) (number of CpG-dinucleotides/100 bp: ICSBP: 10,6;CHI3L1: 2,9; TLR2: 6,2; TLR3: 2,1) were methylated using SssI, subjectedto agarose gel electrophoresis (ethidium bromide staining is shown ascontrol) and directly blotted onto nylon membrane. Membranes werestained using MBD2-Fc, HRP-conjugated anti-human Fc and ECL as describedin Example 3.

FIG. 4: Salt concentration-dependent binding of CpG-methylated to MBD-Fcbeads (A) Schematic presentation of human promoter fragments. Circlesmark the position of CpG-dinucleotides (χ: unmethylated—CPM;  SssImethylated—CCL13, TLR2, CHI3L1). (B, C) A mixture of methylated andun-methylated fragments were bound to MBD2-Fc-sepharose (amount ofMBD2-Fc/50 μl protein A-sepharose is given) eluted using increasing saltconcentrations, purified and separated using agarose gel electrophoresis(along with ⅕ of the Input mixture). Bands were visualised with ethidiumbromide and scanned using a Typhoon Imager (Pharmacia-Amersham).

FIG. 5: Enrichment of CpG-islands by MCIp. Genomic DNA (300 ng) of theindicated cell types was subjected to MCIp. The enrichment of three CpGisland promoters (TLR2, p15 and ESR1) was quantified using LightCyclerreal-time PCR. The amount of a particular promoter fragment amplifiedfrom the MCIp-eluate is shown relative to the untreated genomicDNA-control. The p15 promoter was undetectable in THP-1 cells indicatinga mutation or deletion of this gene.

FIG. 6: Sensitivity of methylated CpG-island detection by MCIp.Decreasing amounts of restricted genomic U937 DNA was subjected to MCIp.The enrichment of the two CpG island promoters (TLR2, p15) wasquantified using LightCycler real-time PCR. The amount of a particularpromoter fragment amplified from the MCIp-eluate is shown relative tothe untreated genomic DNA-control.

FIG. 7: Principle of MB-PCR. This figure shows a schematicrepresentation of MB-PCR.

FIG. 8: MB-PCR of TLR2, ESR1 and p15 promoters in a normal and fourleukemic DNA samples. Genomic DNA (10 ng) of the indicated cell typeswas subjected to MB-PCR. The enrichment of three CpG island promoters(TLR2, p15 and ESR1) was detected by standard genomic PCR. The p15promoter was undetectable in THP-1 cells indicating a mutation ordeletion of this gene.

FIG. 9: MCIp detection of CpG methylation in specific CpG islandpromoters using real-time PCR. (A-C) Fractionated Methyl-CpGimmunoprecipitation (MCIp) was used in combination with real-timeLightCycler PCR to detect the methylation status of the indicated genesfrom untreated (gray bars) and SssI-methylated and MseI-restrictedgenomic DNA fragments (black bars). Recovered gene fragments fromMCIp-eluates (NaCl-concentrations (in mM) are given in boxes above) andan equivalent amount of input-DNA were amplified by LightCycler-PCR.Values (mean±SD, n=4) of individual fractions represent the percentageof recovery and are calculated relative to the amount of PCR-productgenerated from the respective input-DNA (100%). Above each figure a 3 kBregion of the corresponding CpG island is schematically presented. EachCpG dinucleotide is represented by a vertical line. The positions ofexons are indicated as grey boxes and transcription start sites by anarrow. The white box represents a 100 by fragment. Black boxes indicatethe positions of the MseI-fragments that are detected. (D-G) SNRPN,TLR2, ESR1 and CDKN2B gene fragments in the high salt (1000 mM) MCIpfraction of three human myeloid leukaemia cell lines (KG-1, U937 andTHP-1) as well as normal human blood monocytes (N) were analysed by Realtime PCR as above.

FIG. 10: Sensitivity and linearity of the MCIp approach. (A) Decreasingamounts of MseI-treated U937 DNA were subjected to MCIp. CDKN2B and TLR2gene fragments were quantified as above. (B). MseI-treated DNA of normalhuman blood monocytes (N) and KG-1 cells was mixed at the indicatedratios and the mixture was subjected to MCIp and the TLR2 gene fragmentwas quantified using LightCycler-PCR as above.

A better understanding of the present invention and of its manyadvantages will be seen from the following examples, offered forillustrative purposes only, and are not intended to limit the scope ofthe present invention in any way.

EXAMPLE 1 Cloning of pMTBip/MBD2-Fc

A cDNA corresponding to the methyl-CpG binding domain (MBD) of humanMBD2 (Genbank acc. no. NM_(—)003927; AA 144-230) was PCR-amplified fromreverse transcribed human primary macrophage total RNA using primersMBD2-Nhe_S (5′-AGA TGC TAG CAC GGA GAG CGG GAA GAG G-3′) (SEQ ID NO: 4)and MBD2-Not_AS (5′-ATC ACG CGG CCG CCA GAG GAT CGT TTC GCA GTC TC-3′)(SEQ ID NO: 5) and Herculase DNA Polymerase (Stratagene). Cyclingparameters were: 95° C., 3 min denaturation; 95° C., 20 s, 65° C., 20 s,72° C., 80 s amplification for 34 cycles; 72° C., 5 min final extension.The PCR-product was precipitated, digested with Not I/Nhe I, cloned intoNotI/NheI-sites of Signal pIg plus vector (Ingenius, R&D Systems) andsequence verified resulting in pIg/MBD2-Fc (eucaryotic expressionvector). To clone pMTBip/MBD2-Fc for recombinant expression inDrosophila S2 cells, the Apa I/Nhe I—fragment of pIg/MBD2-Fc containingthe MBD of human MBD2 fused to the Fc-tail of human IgG1 was subclonedinto Apa I/Spe I—sites of pMTBiP/V5-His B (Invitrogen).

EXAMPLE 2 Recombinant Expression of an Antibody-LikeMethyl-CpG-DNA-Binding Protein

Methylated Cytosine in single stranded, but not double-stranded DNAmolecules can be efficiently detected using 5-mC antibodies. To enablean antibody-like detection of double-stranded CpG-methylated DNA, avector as described in Example 1, above, was constructed encoding afusion protein comprising the methyl-CpG binding domain (MBD) of humanmethyl-CpG-binding domain 2 (MBD2), a flexible linker polypeptide andthe Fc portion of human IgG1. The protein was expressed under thecontrol of a metal-inducible promoter in Drosophila S2 Schneider-cells,and collected from the supernatant via Protein A affinitychromatography. The purified protein was expressed in high amounts (4-5mg/L cell culture supernatant) and had the expected molecular weight ofappr. 40 kDa (s. FIG. 2).

Accordingly, in detail an insect cell system was chosen for recombinantexpression of MBD2-Fc protein for several reason. The main reason is theabsence or low abundance of CpG-methylation. Production of the proteinin mammalian (especially human) cells may result in DNA contaminations(bound to the MBD2-Fc protein in the cell culture supernatant) which maycomplicate subsequent analysis of CpG-methylated DNA. Other reasonsinclude the simple culture conditions and the potentially high yields ofprotein.

Drosophila S2 cells were obtained from ATTC and cultured inInsect-Xpress medium (Bio Whittaker) containing 10% FCS (PAA) in anincubator at 25° C.

4×10⁶ Drosophila S2 cells/60 mm cell culture dish were transfected witha mixture of 1.5 μg pMTBip/MBD2-Fc and 0.3 μg pCoHygro (Invitrogen)using Effectene transfection reagent (Qiagen) according to themanufacturers protocol. On day three, transfected cells were harvested,washed and replated in selection medium (Insect-Xpress) containing 10%FCS and 300 μg/ml Hygromycin (BD Biosciences). Selection medium wasreplaced every 4-5 days for five weeks. The pool of stably transfectedDrosophila S2 cells was expanded and several aliquots preserved inliquid nitrogen. For large scale production, 1-5×10⁸ cells were culturedin 100-200 ml Insect-Xpress without FCS (optional: 300 μg/ml Hygromycin)in 2000 ml roller bottles for two days before the addition of 0.5 mMCuSO₄. Medium was harvested every 4-7 days and cells were replatedmedium plus CuSO₄ for further protein production. Cell culturesupernatants were combined, dialysed against TBS (pH 7.4) and purifiedusing a protein A column. The MBD-Fc containing fractions were combinedand dialysed against TBS (pH 7.4). The stably transfected Drosophila S2cells produced 3-5 mg recombinant MBD2-Fc protein per litre cell culturesupernatant.

EXAMPLE 3 Detection of CpG-Methylated DNA on Membranes (ReverseSouth-Western Blot)

To test, whether MBD2-Fc was able to detect CpG-methylated DNA onmembrane in a Western blot-like procedure, we blotted in vitromethylated or unmethylated PCR-fragments with different CpG density ontoa Nylon-membrane using a capillary transfer system equivalent totraditional Southern blotting, however without denaturing the DNA priorto blotting. As shown in FIG. 3, using standard immunoblot conditionsand MBD-Fc as an equivalent to the primary antibody, methylated DNA canbe detected on Nylon membranes in a linear fashion (FIG. 3A) anddepending on the CpG content (FIG. 3B). These results indicated that theMBD-Fc fusion protein is able to detect CpG-methylated DNA bound to asolid support.

EXAMPLE 4 Small Scale Enrichment of CpG-Methylated DNA UsingMethyl-CpG-Immunoprecipitation (MCIp)

The following protocol allows a quick enrichment of CpG-methylated DNAfragments using spin columns. The DNA is bound to MBD2-Fc proteincoupled to Sepharose beads via Protein A. The affinity for methylatedDNA increases with the density of methylated CpG-dinucleotides anddecreases with the ionic strength of the wash buffer.

4.1 Binding of the MBD2-Fc Protein to Protein a Sepharose

-   -   8-10 μg purified MBD2-Fc protein was added to 50 μl Protein A        Sepharose 4

Fast Flow beads (Amersham) in 1 ml TBS and rotated over night on arotator at 4° C. On the next day, MBD2-Fc-beads were washed twice withbuffer A (20 mM Tris-HCl pH 8.0, 2 mM MgCl₂, 0.5 mM EDTA, 150 mM NaCl,0.1% NP-40).

4.2 Restriction digest and quantitation of DNA

-   -   At least 1 μg genomic DNA (prepared using Qiagen columns) was        digested using Mse I. Complete digest was controlled using        agarose gel electrophoresis and digested DNA was exactly        quantified using PicoGreen dsDNA Quantitation Reagent (Molecular        Probes).

4.3 Purification of highly methylated CpG-DNA

-   -   Digested DNA (300 ng) was added to the washed MBD2-Fc-beads in 1        ml buffer A and rotated for 3 h on a rotator at 4° C. Beads were        transferred into SpinX-columns and spin-washed with        approximately 1 ml buffer A. Beads were washed twice with 400 μl        buffer B (20 mM Tris-HCl pH 8.0, 2 mM MgCl₂, 0.5 mM EDTA, 450 mM        NaCl, 0.1% NP-40) and twice with buffer C (20 mM Tris-HCl pH        8.0, 2 mM MgCl₂, 0.5 mM EDTA, 650 mM NaCl, 0.1% NP-40). Flow        through of each wash step was either discarded or collected for        further analyses. CpG-methylated DNA was eluted with 250 μl        buffer D (20 mM Tris-HCl pH 8.0, 2 mM MgCl₂, 0.5 mM EDTA, 1000        mM NaCl, 0.1% NP-40) into a new tube. Eluted DNA was desalted        using Qiaquick Spin columns (ELUTED). In parallel, 300 ng        digested DNA (INPUT) was resuspended in 250 μl buffer D and        desalted using QIAquick PCR Purification Kit (Qiagen). Both        ELUTED- and INPUT-DNA was exactly quantified using PicoGreen        dsDNA Quantitation Reagent (Molecular Probes).

4.4. Alternative Approaches

-   -   DNA may be restricted using different restriction endonucleases        or by sonication.

EXAMPLE 5 Detection and Quantitation of Methylated CpG-DNA FragmentsGenerated by MCIp

To test, whether the MBD-Fc fusion protein was able to bindCpG-methylated DNA fragments in an immunoprecipitation-like approach, wefirst tested the binding properties of in vitro generated anddifferentially methylated DNA-fragments. PCR fragments of humanpromoters with varying CpG-density were generated using PCR (s. FIG. 4)and CpG-methylated using SssI (CCL13, TLR2, CHI3L1) or leftun-methylated (CPM). DNA was bound to MBD-Fc-Protein A sepharose beadsin 150 mM NaCl (s. Example 4) and eluted using increasing concentrationsof NaCl. Fractions were collected, spin-purified and subjected toagarose gel electrophoresis. As shown in FIG. 4B, the affinity of amethylated fragment increased with the density of methylatedCpG-dinucleotide, with unmethylated DNA (CPM promoter fragment) elutingat relatively low salt concentrations and highly methylated DNA (TLR2promoter fragment) eluting at high salt concentrations. Variation of theamount of Input-DNA did not significantly change the elution profile.However, the salt-dependent affinity of DNA was dependent on the densityof the MBD-Fc fusion protein on the protein A sepharose beads. Theseresults indicated that the MBD-Fc fusion protein is able to capture andbind CpG-methylated DNA in solution in a salt concentration- andCpG-methylation density-dependent fashion.

5.1 Quantitation on single gene level using gene-specific Real-time PCR

-   5.1.1 To test whether the recombinant MBD-Fc protein was able to    detect the methylation density of a CpG island promoter in a complex    genomic DNA mixture, genomic DNA from three leukemia cell lines,    normal donor monocytes as well as blast cells from a patient with    AML were restricted with Mse I and subjected to MCIp. The enrichment    of three CpG island promoters (TLR2, p15 and ESR1) in the 1000 mM    NaCl MCIp-fraction was detected using LightCycler-PCR. The three    loci were chosen because p15 and ESR1 are known targets for    methylation in leukemia and TLR2 was previously shown to be    methylated in U937 cells, but not in THP-1 cells. As shown in FIG.    5, none of the three loci was significantly detectable in the DNA    preparation from the normal donor DNA (MO), which is consistent with    a usually unmethylated state of CpG island promoters in normal    cells. The enrichment of TLR2 in U937 but not in THP-1 is consistent    with the previously observed methylation pattern in both cells.    Bisulfite sequencing of the TLR2 promoter as described in Hähnel, J.    Immunol. 168 (2002), 5629-37) demonstrated an almost complete    methylation of the TLR2 promoter in KG1-cells (data not shown) which    is consistent with the strong MCIp-enrichment shown in FIG. 5. The    results for p15 in KG1 and U937 are consistent with published data.    These data indicate that MCIp can be used to detect methylated DNA    fragments of single gene fragments in genomic DNA.

Accordingly, enrichment of a specific Mse I-fragment in the MCIp eluatewas detected and quantified relative to the genomic INPUT by Real-timeLightcycler-PCR. (s. FIG. 5). The enrichment may also be quantifiedafter an unspecific DNA-amplification of both ELUTED- and INPUT-DNA (s.amplicon generation in Example 5.2.1 below, data not shown).

TABLE 1-1 Gene-specific oligonucleotide primers for CpG-island promotersMse I fragment product Gene (bp) Sense primer Antisense primer (bp) TLR21358 TGTGTTTCAGGTGATGTGAGGTC CGAATCGAGACGCTAGAGGC 118 (SEQ ID NO: 6)(SEQ ID NO: 7) p15 699 GGCTCAGCTTCATTACCCTCC AAAGCCCGGAGCTAACGAC 87 (SEQID NO: 8) (SEQ ID NO: 9) ESR1 1108 GACTGCACTTGCTCCCGTCAAGAGCACAGCCCGAGGTTAG 129 (SEQ ID NO: 10) (SEQ ID NO: 11)

In order to test whether MCIp may be used to discriminate methylated andunmethylated DNA fragments from genomic DNA, MCIp was used to enrichMseI-restricted genomic DNA of in vitro SssI-methylated and untreatednormal DNA from monocytes of a healthy donor. MseI was chosen for DNAfragmentation, because it is known to preferentially cut in regions oflow CpG content while leaving many CpG islands uncut (Cross, Nat. Genet.6 (1994), 236-244).

The salt concentration-dependent enrichment of four different CpG-islandpromoters and a promoter with low CpG density was determined inSssI-methylated and untreated DNA relative to the input-DNA usingLightCycler real-time PCR. As a positive control for DNA methylation,the SNRPN gene promoter that is subject to maternal imprinting with oneof its two copies being methylated also in normal cells (Zeschnigk, Hum.Mol. Genet. 6 (1997), 387-395) was used. In normal DNA the twodifferentially methylated allele-fragments of SNRPN were enriched in twoseparate fractions (s. FIG. 9A). Only one enriched fraction was observedwith SssI-methylated DNA. In the case of CDKN2B gene (also known asp15^(INK4b)) which is known to be frequently methylated in leukaemiacells (Chim, Ann. Hematol. 82 (2003), 738-742; Dodge, Int. J. Cancer 78(1998), 561-567; Dodge, Leuk. Res. 25 (2001), 917-925) (FIG. 9B), thefragment was detected mainly in a low salt fraction from normal DNA andin the high salt fraction from SssI-methylated DNA. Similar results wereobtained for the human estrogen receptor 1 (ESR1) gene (Issa, CancerRes. 56 (1996), 973-977) and the human Toll-like receptor 2 gene (TLR2)(data not show). As shown in FIG. 9C, the profiles of methylated andunmethylated DNA at the CHI3L1 locus were significantly different fromthose of the above tested CpG island promoters. Most of the untreatedCHI3L1-fragment was recovered at lower NaCl concentrations, and a slightshift was observed towards higher NaCl concentrations when the DNA wasSssI-methylated. Analysis of the above elution profiles suggests that:

-   a.) A two to three hundred-fold enrichment of stronger over less    methylated genomic fragments can be obtained in either low or high    salt fractions;-   b.) Fragments with low CpG density are largely excluded from the    high salt fraction.-   c.) The fractionated MCIp approach allows the resolution of small    differences in CpG methylation density (the average difference    between SssI-treated and untreated monocyte DNA is approximately six    out of twelve methylated CpG residues, data not shown);

In order to test whether MCIp can detect aberrant hypermethylation intumor samples, DNA from three leukaemia cell lines (KG1, U937, THP-1) aswell as from monocytes of a healthy donor were analyzed for SNRNP,CDKN2B, ESR1 and TLR2 promoter enrichment in the high salt fraction (s.FIG. 9D-G). The TLR2 gene promoter was enriched in KG-1 and U937 cells,but not in THP-1 or normal cells. The methylation pattern of TLR2 wasconfirmed by bisulfite sequencing (Haehnel, J. Immunol. 168 (2002),5629-5637) (data not shown). Results for CDKN2B (KG-1 and U937) and ESR1(KG-1) were also in line with previously published studies (Chim (2003);Dodge (2001); Issa (1996), all loc. cit.). None of the above three MseIfragments was significantly enriched in the DNA from normal cells. Inconcordance with its imprinting-related methylation status the SNRPNgene promoter was significantly enriched in all leukaemia cell lines aswell as in normal cells. These experiments established that the highsalt MCIp fraction specifically enriches genomic DNA-fragments with ahigh degree of CpG methylation.

TABLE 1-2 Gene-specific oligonucleotide primers for real- timeamplification of CpG-island promoters Gene Primer sequence (sense &antisense) SNRNP 5′-TAC ATC AGG GTG ATT GCA GTT CC-3′ (SEQ ID NO: 12)5′-TAC CGA TCA CTT CAC GTA CCT TCG-3′ (SEQ ID NO: 13) TLR2 5′-TGT GTTTCA GGT GAT GTG AGG TC-3′ (SEQ ID NO: 14) 5′-CGA ATC GAG ACG CTA GAGGC-3 (SEQ ID NO: 15)′ ESR1 5′-GAC TGC ACT TGC TCC CGT C-3′ (SEQ ID NO:16) 5′-AAG AGC ACA GCC CGA GGT TAG-3′ (SEQ ID NO: 17) CDKN2B 5′-GGC TCAGCT TCA TTA CCC TCC-3′ (SEQ ID NO: 18) 5′-AAA GCC CGG AGC TAA CGA C-3′(SEQ ID NO: 19) CHI3L1 5′-ATC ACC CTA GTG GCT CTT CTG C-3′ (SEQ ID NO:20) 5′-CTT TTA TGG GAA CTG AGC TAT GTG TC-3′ (SEQ ID NO: 21)

-   5.1.2 In order to determine the amount of DNA required for the    detection of a single gene fragment in a complex mixture of genomic    DNA, decreasing amounts of DNA fragments were subjected to MCIp and    subsequent LightCycler real-time PCR. As shown in FIG. 6, the    methylated TLR2 promoter can be enriched and detected from as little    as 1 ng genomic DNA from U937 cells. The un-methylated p15-promoter    was not significantly enriched (20 ng MCIp-eluate) or not detectable    (4 ng or 1 ng MCIp-eluate) in U937 cells (FIG. 6). These results    indicate that MCIp is a sensitive method to detect methylated    DNA-fragments in a complex genomic mixture.-    In order to test the sensitivity of the approach, decreasing    amounts of U937 DNA were analyzed using the MCIp approach. The    enrichment of TLR2 (strong methylation) and CDKN2B gene fragments    (no methylation) were determined by LightCycler real-time PCR. As    shown in FIG. 10A, a significant enrichment of the TLR2 fragment was    achieved using as little as 1 ng of genomic DNA fragments    (equivalent to approximately 150 tumor cells) for the MCIp    procedure. Samples derived from tumors may contain significant    numbers of normal cells, that would be expected to be unmethylated    at most CpG islands. To test how linear the detection of CpG    methylation is with respect to cell purity, MCIp was performed using    mixtures of DNA from normal blood cells and the leukaemia cell line    KG-1 showing high levels of CpG island methylation at several    promoters. As shown in FIG. 10B, the TLR2 promoter fragment was only    detected in samples containing KG-1 DNA and the signal gradually    increased with the proportion of methylated DNA in the sample.    Similar results were obtained for the ESR1 locus (data not shown).    In general, most informative (with respect to effects on    transcription) and clearest results (in terms of noise and    background) were obtained when a target gene fragment contained only    the proximal promoter within the CpG island. Also, in addition to    enzyme restriction, DNA fragmentation may also be achieved by    mechanical means, e.g. sonication (data not shown).

5.2 Quantitation on Genome-Wide Level Using Microarray Technology

5.2.1 Generation of DNA-Amplicons from Genomic Mse I-Fragments UsingLigation-Mediated (Lm)-PCR

-   -   To generate a Mse 1-compatible LMPCR-Linker, oligonucleotides        LMPCR_S-L (5′-GCG GTG ACC CGG GAG ATC TCT TAA G-3′) (SEQ ID        NO: 22) and LMPCR_AS-L (5′-TAC TTA AGA GAT C-3′) (SEQ ID NO: 23)        were annealed as follows. Both oligos were combined at a        concentration of 20 μM in nuclease-free H₂O (USB), incubated at        80° C. for 10 min, and cooled down slowly to RT. The annealed        Linker was stored in 50 μl-aliquots at −20° C.    -   LMPCR-Linker (0.5 μl/ng ELUTED- or INPUT-DNA) was ligated to the        ELUTED- and in a separate reaction to an equal amount of        INPUT-DNA in 60 μl reactions using 1 μl T4-Ligase (1200 u/μl,        NEB) at 16° C. o/n. Linker-ligated DNA was desalted using        QIAquick PCR Purification Kit (Qiagen) and eluted in 55 μl        Tris-HCl pH 8.0 (5 mM).    -   Linker-ligated DNA (ELUTED- and INPUT separately) was        PCR-amplified using LMPCR-Primer (5′-GTG ACC CGG GAG ATC TCT TAA        G-3′) (SEQ ID NO: 24) and Taq DNA Polymerase (Roche). The PCR        mix contained 25 μl 10× PCR-buffer (Roche), 15 μl MgCl₂ (25 mM,        Roche), 10 μl dNTPs (10 mM each) 65 μl Betain (5M, Sigma), 2.5        μl LMPCR-Primer, 45 μl of linker-ligated DNA, 2.5 μl Taq DNA        Polymerase (5 U/μl) in a total volume of 250 μl which was        distributed into five PCR-tubes. Cycling parameters were: 58°        C., 2 min (melting off LMPCRAS-L), 72° C. 5 min (fill in        overhangs); 95° C., 30 s, 58° C., 30 s, 72° C., 3 min        amplification for 15 cycles; 72° C., 10 min final extension.    -   PCR-Reactions were combined and purified using QIAquick PCR        Purification Kit (Qiagen). Both ELUTED- and INPUT-amplicons were        exactly quantified using PicoGreen dsDNA Quantitation Reagent        (Molecular Probes).

5.2.2 Analysis of MCIp-Amplicons Using CpG-Island Microarrays

-   -   MCIp-Amplicons may be analysed using PCR (LightCycler, Standard        PCR) to detect the enrichment of single gene fragments. To        detect multiple gene fragments array technology may be used. The        analysis of MCIp-amplicons using for example CpG island        microarrays will involve the fluorescent labelling of        MCIp-DNA-fragments and subsequent hybridization to microarrays        using standard protocols.

EXAMPLE 6 Single-Tube Assay for the Detection of CpG-MethylatedDNA-Fragments Using Methyl-Binding Polymerase Chain Reaction (MB-PCR)

This method uses an approach similar to ELISAs. A protein with highaffinity for CpG-methylated DNA is coated onto the walls of a PCR-cyclercompatible reaction vessel and used to selectively capture stronglymethylated DNA-fragments from a genomic DNA mixture. The retention of aspecific DNA-fragment (e.g. a CpG island promoter of a specific gene)can be detected in the same tube using PCR (either standard PCR orrealtime PCR, single or multiplex). The degree of methylation may beestimated relative to a PCR reaction of the genomic input DNA. FIG. 7shows a schematic representation of MB-PCR.

6.1 DNA Preparation and Fragmentation

-   -   Genomic DNA from three cell lines (KG1, U937, and THP-1), normal        human monocytes (healthy donor) and frozen blast cells from a        patient with AML were prepared using Blood and Cell Culture Midi        Kit (Qiagen). Quality of the genomic DNA-preparation was        controlled by agarose gel electrophoresis and DNA concentration        was determined by UV spectrophotometry. Genomic DNA was digested        with Mse I (NEB) and finally quantified using PicoGreen dsDNA        Quantitation Reagent (Molecular Probes).

6.2 Preparation of PCR Tubes

-   -   MBD-Fc-coated PCR tubes were prepared using heat stable        TopYield™ Strips (Nunc Cat. No. 248909). 50 μl of recombinant        MBD-Fc protein (diluted at 15 μg/ml in 10 mM Tris/HCl pH 7.5)        were added to each well and incubated overnight at 4° C. Wells        were washed three times with 200 μl TBS (20 mM Tris, pH 7.4        containing 150 mM NaCl) and blocked overnight at 4° C. with 100        μl Blocking Solution (10 mM Tris, pH 7.5 containing 150 mM NaCl,        4.5% skim milk powder, 5 mM EDTA and 0.8 μg/ml of each poly        d(I/C), poly d(A/T and poly d(CG)). Tubes were washed three        times with 200 μl TBST (TBS containing 0.1% Tween-20.

6.3 Binding of Methylated DNA

-   -   50 μl Binding Buffer (20 mM Tris, pH 7.5 containing 400 mM NaCl,        2 mM MgCl₂, 0.5 mM EDTA, and 0.1% Tween-20) were added to each        well and 1 μl Mse I-digested DNA (10 ng/μl) was added to every        second well (M-reaction). Wells were incubated on a shaker at        4° C. for 3 hours. Tubes were washed three times with 200 μl        Binding Buffer and once with 10 mM Tris/HCl pH 7.5.

6.4 Detection of Methylated DNA Fragments

-   -   PCR was carried out directly in the TopYield™ Strips. The        PCR-Mix (50 μl/well) contained a standard PCR buffer (Roche),        2.5 U FastStart Taq DNA Polymerase (Roche), 10 pmol of each        gene-specific primer (synthesized by Qiagen), dNTPs (200 mM        each, Amersham/Pharmacia) 1 M betaine (Sigma), primer sequences        and cycling parameters are shown in Table 2 & 3, respectively.        After adding the PCR-mix, 1 μl Mse I-digested DNA (10 ng/μl) was        added to every second other well, that was not previously        incubated with DNA-fragments (P-reaction). PCR-products were        analysed using agarose gel electrophoresis and the ethidium        bromide stained gel was scanned using a Typhoon 9200 Imager        (Amersham/Pharmacia).

TABLE 2 Cycling parameters (MB-PCR): 94° C. 3 min 94° C. 30 s 60° C. 30s 37 x 72° C. 50 s 72° C. 5 min 15° C. ∞

TABLE 3 Gene-specific oligonucleotide primers for CpG-island promotersMse I fragment product Gene (bp) Sense primer Antisense primer (bp) TLR21358 TGTGTTTCAGGTGATGTGAGGTC CGAATCGAGACGCTAGAGGC 118 (SEQ ID NO: 14(SEQ ID NO: 15) p15 699 GGCTCAGCTTCATTACCCTCC AAAGCCCGGAGCTAACGAC 87(SEQ ID NO: 8) (SEQ ID NO: 9) ESR1 1108 GACTGCACTTGCTCCCGTCAAGAGCACAGCCCGAGGTTAG 129 (SEQ ID NO: 16) (SEQ ID NO: 17)

FIG. 8 shows the result of an MB-PCR experiment analysing themethylation profile of three different CpG-island promoters in five celltypes. The lanes marked with P represent the amplification of thegenomic input DNA. With an exception of the (probably deleted ormutated) p15 gene in THP-1 cells, all promoters were amplified. Notably,none of the promoters was detected in the MB-PCR reactions from thenormal DNA control, which is consistent with the fact that thesepromoters are not methylated in normal individuals. In the cell lines aswell as in the patient sample, promoters were mostly methylated. Theresults correspond to the data obtained with MCIp in independentexperiments.

1. A reagent for binding methylated DNA comprising: a first polypeptideand a second polypeptide each comprising: (i) a methyl-DNA-bindingdomain of an MBD2 protein; (ii) an Fc portion of an antibody and (iii) aflexible peptide linker, wherein the first polypeptide and secondpolypeptide each have the methyl-DNA-binding domain of the MBD2 proteinfused to the Fc portion of an antibody through the flexible peptidelinker and the Fc portion of an antibody of the first polypeptide isbonded to the Fc portion of an antibody of the second polypeptide; and abivalent binding site for methylated DNA comprising themethyl-DNA-binding domain of the first polypeptide in proximity to themethyl-DNA-binding domain of the second polypeptide, wherein thebivalent binding site can bind two methylated CpG residues that are onthe same strand of a double stranded DNA molecule or on differentstrands of a double stranded DNA molecule.
 2. The reagent of claim 1,wherein the capacity to bind to methylated DNA is dependent on thedegree of methylation.
 3. The reagent of claim 1, wherein the capacityto bind to methylated DNA is dependent on salt concentration.
 4. Thereagent of claim 1, wherein the MBD2 protein is a human MBD2 protein. 5.The reagent of claim 1, wherein the reagent can detect methylated DNA ina sample of less than 10 ng of genomic DNA.
 6. The reagent of claim 1,wherein the reagent can detect methylated DNA in a sample of less than 5ng of genomic DNA.
 7. The reagent of claim 1, wherein the reagent candetect methylated DNA in a sample of less than 1 ng of genomic DNA.
 8. Acomposition comprising the reagent of claim 1.